Bio-Rad Bio-Dot User Manual
Contents
1. A rtisan Artisan Technology Group is your source for quality TecmoogyGroup new and certified used pre owned equipment FAST SHIPPING AND SERVICE CENTER REPAIRS WE BUY USED EQUIPMENT DELIVERY Experienced engineers and technicians on staff Sell your excess underutilized and idle used equipment TENS OF THOUSANDS OF at our full service in house repair center We also offer credit for buy backs and trade ins IN STOCK ITEMS www artisantg com WeBuyEquipment 7 EQUIPMENT DEMOS HUNDREDS OF InstraV ea REMOTE INSPECTION LOOKING FOR MORE INFORMATION MANUFACTURERS Remotely inspect equipment before purchasing with Visit us on the web at www artisantg com 7 for more our interactive website at www instraview com information on price quotations drivers technical LEASING MONTHLY specifications manuals and documentation RENTALS ITAR CERTIFIED D a gaa tia Contact us 888 88 SOURCE sales artisantg com www artisantg com Bio Dot Microfiltration Apparatus Instruction Manual Catalog Numbers 170 6545 170 6547 For technical service call your local Bio Rad office or in the U S call 1 800 4BIORAD 1 800 424 6723 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Section 1 1 1 Section 2 2 1 2 2 Section 3 3 1 3 2 Section 4 4 1 4 2 Section 5 Section 6 6 1 6 2 Section 7 7 1 7 2 7 3 7 4 Section 8 8 1 8 2 Section 9 Sect2. Immediately before use fragment and denature the probe and carrier DNA as follows Add to the precipitated RNA probe 0 1 ml of yeast RNA 20 mg ml 0 5 ml of carrier DNA 10 mg ml and 0 6 ml of deionized formamide mix thoroughly and heat at 70 C for 5 minutes 2 Cut one corner of the bag remove the prehybridization solution and replace it with hybridization solution DNA Bound to DNA Bound to Zeta Probe Membrane Nitrocellulose 50 formamide 50 formamide 1 5x SSPE 1x Denhardt s solution 1 SDS 0 1 SDS 0 5 nonfat dry milk 100 ug ml denatured salmon sperm DNA 3 Add probe then seal the open corner taking care to exclude all air bubbles Mix the contents of the bag thoroughly Incubate at 50 C for 4 24 hours Note After beginning hybridization the membranes should not be permitted to dry Washes 1 At the completion of hybridization remove the membranes from their hybridization bags into 2x SSC Rinse briefly then wash them sequentially with agitation for 15 minutes at room temperature in the following solutions a 2x SSC 0 1 SDS b 0 5x SSC 0 1 SDS C 0 1x SSC 0 1 SDS 2 For DNA bound to nitrocellulose membranes it may be necessary to include an RNase treatment in the wash Treat membranes with 20 ig ml RNase for 30 minutes at 37 C in 2x SSC Sanizen et al 1986 3 After washing the blotted membranes are ready for autoradiography If no further cycles of hybridization are to be done on the membrane then the membran
3. Formamide Dilute 50 0 g formamide to 100 ml with ddH O Store at 4 C Immediately before use deionize the required volume by stirring gently for 1 hour with 1 g mixed bed ion exchange resin AG 501 X8 D resin catalog number 142 6425 per 10 ml of formamide Filter through coarse filter paper For DNA or RNA Bound to For DNA Bound to For RNA Bound to Zeta Probe Membrane Nitrocellulose Nitrocellulose See instruction manual Maniatis et al 1982 Thomas 1980 1 mM EDTA 6x SSC 50 formamide 7 SDS 0 5 SDS 5x SSC 0 5 M NaHPO pH 7 2 5x Denhardt s solution 1x Denhardt s solution 100 ug ml denatured 50 mM NaHPO pH 6 5 salmon sperm DNA 250 ug ml denatured 1 mM EDTA salmon sperm DNA For DNA or RNA Bound to For DNA Bound to For RNA Bound to Zeta Probe Membrane Nitrocellulose Nitrocellulose A Wash 2 times for A Rinse in A Wash 4 times at room 30 60 minutes at 65 C in 2x SSC 0 5 SDS temperature for 5 minutes 1 mM EDTA in 2x SSC 0 1 SDS 40 mM NaHPO pH 7 2 5 SDS B Wash 2 times for B Wash at room B Wash 2 times at 30 60 minutes at 65 C in temperature for 50 C in 1 mM EDTA 5 minutes in 2x SSC 0 5 SDS 0 1x SSC 0 1 SDS 0 5x SSC 0 1 SDS 1mM EDTA 40 mM NaHPO pH 7 2 1mM EDTA 1 SDS 17 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com DNA Bound to DNA Bound to Zeta Probe Membrane Nitrocellulose 50 formamide 50 formamide 1 5x SSPE 1x Denhardt s solut
4. or by gravity filtration Notes a method for applying gentle vacuum to the apparatus is to adjust the flow valve to setting 3 Use a finger to cover the valve port exposed to air The amount of vacuum reaching the manifold will be regulated by the pressure of your finger on the valve 6 After the sample has filtered through add 500 ul 0 4 M NaOH to each well for Zeta Probe membrane or 2x SSC for nitrocellulose Apply the vacuum by setting the 3 way valve to setting 1 until the sample wells are empty 7 Disassemble the Bio Dot Apparatus Remove the blotted membrane and rinse it in 2x SSC Allow the membrane to air dry The Zeta Probe membrane is ready for hybridization immediately after air drying If hybridization is not to be undertaken within 2 days then vacuum bake the blotted Zeta Probe membrane at 80 C for 30 minutes Nitrocellulose membrane must be baked under vacuum for 2 hours at 80 C before hybridization The Zeta Probe membrane and nitrocellulose membranes can be stored dry between two pieces of filter paper in plastic bags at 23 25 C Section 6 RNA Blotting RNA must be denatured prior to application to Zeta Probe or nitrocellulose membranes to ensure optimal hybridization Two protocols are presented for denaturing RNA samples Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 6 1 Alkaline RNA Denaturation and Fixation 1 Always wear gloves when handling blottin
5. 2 L with ddH O Tween Tris Buffered Saline 1x TTBS 1 L 20 mM Tris pH 7 5 500 mM NaCl 0 05 Tween 20 Add 0 5 ml Tween 20 to 1 L of TBS Blocking Solution 100 ml 1 BSA TBS Add 1 0 g bovine serum albumin BSA to 100 ml TBS Stir to dissolve Antibody Buffer 200 ml 1 BSA TTBS Add 2 g BSA to 200 ml TTBS Stir to dissolve 100 ml should be reserved for primary antibody and an equal volume for dilution of the secondary antibody conjugate Primary Antibody Solution 100 ml Dilute antigen specific primary antibody to the appropriate titer in 100 ml of antibody buffer Secondary Antibody Solution 100 ml Dilute species specific Bio Rad secondary antibody conjugate 1 3 000 by adding 33 ul of conjugate to 100 ml of antibody buffer Color Development Solution The specific chemicals and buffers are dependent on the enzyme conjugate being used See the Immun Blot assay kit instruction manual for details on how to make the appropriate solution 8 2 Solutions for Zeta Probe Membrane for Protein Applications When immobilizing antigen onto the Zeta Probe membrane the immunoassay must be performed in a separate container following removal of the membrane from the Bio Dot apparatus Two methods of blocking are given Method A uses nonfat dry milk Jerome and Jiehning 1986 Johnson et al 1984 as the blocking agent Method B uses gelatin and 1 methyl 2 pyrrolidinone MPO as the blocking agents The solutions for the two methods ar
6. addition to the Bio Dot SF slot format microfiltration apparatus Conversion of the Bio Dot apparatus to the Bio Dot SF apparatus is accomplished by purchasing the Bio Dot SF module which provides the 48 well slot format sample template The Bio Dot apparatus is simple to operate As shown in Figure 1 a sheet of membrane is clamped between the gasket and the 96 well sample template The gasket is aligned above the support plate which is placed over the vacuum reservoir This assembly is attached to a vacuum source by the in line 3 way flow valve which allows on off control of vacuum during assay procedures The entire assembly is held together by the four screws on the sample template and the patented rubber sealing gasket seals prevent well to well leakage whether the vacuum is on or off Sample can be easily applied to the 96 well format with a standard pipet or with a Costar Octapette pipet The material used in the construction of the Bio Dot blotting apparatus can withstand rigorous sterilization and cleanup procedures The Bio Dot apparatus can be repeatedly autoclaved and is resistant to many chemicals including acids bases and ethanol 1 1 Specifications Materials Bio Dot apparatus Molded polysulfone Bio Dot gasket Silicone rubber Stopcock Teflon Tubing Tygon Shipping weight 600 g Overall size 13 x 15 x 6 cm Membrane size 12 x 9 cm sheet Autoclaving 15 minutes at 250 F 121 C with a 1 minute fast exhaust Chemical compatibi
7. stabilities For color development in the apparatus the unit is compatible with both the methanol used in horseradish peroxidase HRP color development systems and the low concentration of dimethyl formamide DMF used to solubilize the alkaline phosphatase AP color development reagents However high concentrations of DMF will attack the plastic Also the unit is completely compatible with the low concentrations of diethyl pyrocarbonate DEPC used as an alternative to autoclaving for elimination of RNase activity Table 1 Chemical Compatibility Chemicals compatible with Bio Dot apparatus Hydrochloric acid Methanol Sulfuric acid Ethanol Phosphoric acid Butanol Glacial acetic acid Isopropyl alcohol Sodium hydroxide Formaldehyde Potassium hydroxide Hydrogen peroxide Ammonium hydroxide Ethylene glycol Heptane 5 acetone in H O Nitric acid Chemicals incompatible with Bio Dot apparatus use voids warranty Ethyl acetate Toluene Butyl acetate Benzene Acetone Methyl ethyl ketone Chloroform Methylene chloride Trichloroacetic acid Section 3 Bio Dot Assembly 3 1 Assembly 1 Clean and dry the Bio Dot apparatus and gasket prior to assembly 2 Place the gasket support plate into position in the vacuum manifold There is only one way to slide the plate into the manifold 3 Place the sealing gasket on top of the gasket support plate The guide pins on the vacuum manifold help align the 96 holes in the gasket over the 96
8. water per well for Zeta Probe membranes Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 3 Adjust the flow valve so that the vacuum chamber is open to air flow valve setting 2 Figure 3 Fill the appropriate wells with antigen protein solution using any volume up to 500 ul per well Multiple applications of antigen to a sample well are possible but the most rapid and efficient use of the apparatus is achieved by applying the required amount of antigen in a minimal sample volume Allow the entire sample to filter through the membrane by gravity flow Make sure that the flow valve is positioned at a level below the sample wells to ensure proper drainage during filtration applications This passive filtration is necessary for quantitative antigen binding Each well should be filled with the same volume of sample solution to ensure homogenous filtration of all sample wells Generally it takes 30 40 minutes for 100 ul of the antigen solution to filter through the membrane If antigen is very dilute and it is necessary to ensure that all proteins in the applied sample are filtered through the membrane an optional wash step can be performed To perform this wash add an aliquot of TBS equal to the original sample volume to each sample well Allow this material to passively filter through the membrane by gravity filtration If the membrane is going to be removed from the apparatus following
9. 1977 For alternative protocols see the Zeta Probe membrane instruction manual 2 Add the denatured probe remove all air bubbles and reseal the bag Mix the contents of the bag Hybridize with agitation For DNA or RNA Bound to For DNA Bound to For RNA Bound to Zeta Probe Membrane Nitrocellulose Nitrocellulose 4 24 hours at 65 C 4 24 hours at 68 C 4 24 hours at 42 C 3 Carefully remove the hybridization solution by cutting one corner Remove hybridized Zeta Probe membrane from the plastic bag Note Once hybridization has begun do not let the membrane dry 12 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Washes 1 Agitate the solutions when washing membranes For DNA or RNA Bound to For DNA Bound to For RNA Bound to Zeta Probe Membrane Nitrocellulose Nitrocellulose A Wash 2 times for 30 60 A Rinse in A Wash 4 times at room minutes at 65 C in 2x SSC 0 5 SDS temperature for 5 minutes 1 mM EDTA in 2x SSC 0 1 SDS 40 mM NaHPO pH 7 2 5 SDS B Wash 2 times for 30 60 B Wash at room B Wash 2 times at minutes at 65 C in temperature for 50 C in 1 mM EDTA 5 minutes in 2x SSC 0 5 SDS 0 1x SSC 0 1 SDS 0 5x SSC 0 1 SDS 1mM EDTA 40 mM NaHPO pH 7 2 1 mM EDTA 1 SDS After washing the blotted membrane is ready for autoradiography If no further cycles of hybridization are to be done on the membrane the membrane can be dried When reprobi
10. 7 Chen CW and Thomas CA Jr Recovery of DNA segments from agarose gels Anal Biochem 101 339 341 1980 Cleveland PH et al Rapid and efficient immobilization of soluble and small particulate antigens for solid phase radioimmunoassays J Immunoassay 2 117 136 1981 Cunningham M Spot blot a hybridization assay for specific DNA sequences in multiple samples Anal Biochem 128 415 421 1983 Faulstich H et al Alpha and beta galactosidases bound to nylon nets FEBS Lett 48 226 229 1974 Gershoni JM and Palade GE Protein blotting principles and applications Anal Biochem 131 1 15 1983 Harpold MM et al Construction and identification by positive hybridization translation of a bacterial plasmid containing a rat growth hormone structural gene sequence Nucleic Acids Res 5 2039 2053 1978 Hawkes R et al A dot immunobinding assay for monoclonal and other antibodies Anal Biochem 119 142 147 1982 22 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Herbrink P et al The antigen spot test AST a highly sensitive assay for the detection of antibodies J Immunol Methods 48 293 298 1982 Holland LJ and Wangh LJ Efficient recovery of functionally intact mRNA from agarose gels via trans fer to an ion exchange membrane Nucleic Acids Res 11 3283 3300 1983 Horejsi V and Hilgert Nitrocellulose membrane as an antigen or antibody carrier for screening hyb
11. 9 135 1979 Lin S and Riggs AD The general affinity of lac repressor for E coli DNA implications for gene regulation in procaryotes and eukaryotes 4 107 111 1975 Locker D Motta G Detection of antibody secreting hybridomas with diazobenzyloxymethyl paper an easy sensitive and versatile assay J Immunol Methods 59 269 275 1983 Lye DJ and Birge EA The use of nitrocellulose filters to study DNA binding proteins in crude cell lysates Effect of competing DNA Curr Microbiol 6 139 143 1981 Maniatis T et al Molecular Cloning A Laboratory Manual 1st edn Cold Spring Harbor Laboratory Press Cold Spring Harbor NY 1982 Nakamura K et al Microassay for proteins on nitrocellulose filter using protein dye staining procedure Anal Biochem 148 311 319 1985 Neuhoff V et al Spot analysis for glycoprotein determination in the nanogram range Hoppe Seylers Z Physiol Chem 362 1427 1434 1981 Olmsted JB Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples J Biol Chem 256 11955 11957 1981 Palfree RG and Elliott BE An enzyme linked immunosorbent assay ELISA for detergent solubilized la glycoproteins using nitrocellulose membrane discs J Immunol Methods 52 395 408 1982 Ricciardi RP et al Purification and mapping of specific mRNAs by hybridization selection and cell free translation Proc Natl Acad Sci USA 76 4927 4931 1979 Richman DD et al A rapid radioimmun
12. Open the flow valve to the atmosphere and add 200 400 ul of TTBS wash solution to each well Apply vacuum until the wash solution is drained from the wells Repeat for a total of three wash cycles With the vacuum off and the flow valve open to air add 100 ui of secondary antibody solution to each well Allow gravity filtration to occur 30 40 minutes until all solution has drained from the wells Turn the vacuum on and drain the wells Add 200 400 ul of TTBS wash solution to each well and drain completely Repeat for a total of two washes Note At this point the membrane is ready for development Color development of enzyme conjugated antibodies can be performed in the apparatus or in a separate reservoir If performing autoradiography remove the membrane dry it on a filter paper wrap it with plastic wrap and expose it to X ray film The best method to remove the membrane from the Bio Dot apparatus is to leave the vacuum on following the last wash step While the vacuum is on loosen the screws and remove the sample template Turn off the vacuum and remove the membrane 7 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 12 For color development in a separate vessel remove the membrane and place it in the color development vessel Wash the membrane twice with TBS to remove excess Tween 20 Prepare the color development solution and incubate the membrane in the soluti
13. ays then bake the Zeta Probe membrane under vacuum for 30 minutes at 80 C The Zeta Probe membrane and nitrocellulose membranes can be stored dry between two pieces of filter paper in plastic bags at 23 25 C 6 2 Glyoxal RNA Denaturation and Fixation 1 Prepare RNA samples to the following final concentrations 50 dimethyl sulfoxide DMSO 10 mM NaH PO pH 7 0 1 M glyoxal Incubate the RNA for 1 hour at 50 C Cool the samples on ice 3 Always wear gloves when handling blotting membranes Prewet the blotting membrane by placing it gently at a 45 angle into a tray of wetting solution Wet the Zeta Probe membrane in distilled water nitrocellulose in 6x SSC see Section 9 for solution preparation Assemble the Bio Dot apparatus according to the instructions in Section 3 1 Remember to apply the vacuum and then retighten the screws that hold the apparatus together Samples and wash solutions may be applied with a standard pipet or a Costar Octapette pipet Apply the denatured RNA and pull the sample through by passive filtration or by applying a gentle vacuum Note a method for applying gentle vacuum to the apparatus is to adjust the flow rate valve to setting 3 Use a finger to cover the valve port exposed to air The amount of 10 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com vacuum reaching the manifold will be regulated by the pressure of your finger
14. binding of antigen proceed to step 6 and follow the instructions for the wash step The wash step should be performed prior to disassembling the apparatus to ensure that all antigen is removed from the drain ports underneath the membrane After the antigen samples have completely drained from the apparatus add 200 300 ul of the blocking solution to each well Allow gravity filtration to occur until the blocking solution has completely drained from every well This step should take approximately 60 minutes Do not apply vacuum to speed up this step as it will lead to poor assay results Adjust the flow valve so that the vacuum chamber is exposed to air Add 200 400 ul of the Tween Tris buffered saline TTBS wash solution to each well Adjust the flow valve to the vacuum position and pull the wash solution through the membrane Disconnect the vacuum as soon as the wash solution has drained from all the sample wells Repeat the wash step If the membrane is to be removed from the apparatus prior to performing an immunoassay remove it at this point Otherwise proceed to step 7 Note For better results with Zeta Probe use 0 3 Tween 20 Open the flow valve to air Add 100 ul of primary antibody solution to each sample well Allow gravity filtration to occur until the antibody solution has completely drained from the sample wells approximately 30 40 minutes Apply vacuum to the apparatus to remove any excess liquid from the sample wells
15. brane following the vacuum procedure in step 7 If this step is not performed prior to applying samples assay results will show halos or weak detection signal 9 Gently remove the buffer from the wells by vacuum flow valve setting 3 Figure 3 Watch the sample wells As soon as the buffer solution drains from all the wells adjust the flow valve so that the unit is exposed to air and disconnect the vacuum At this point the unit is ready for sample application Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Flow valve setting 1 The vacuum manifold is exposed to the vacuum source only Use for applying vacuum to the Bio Dot apparatus Bio Dot Vacuum Air Flow valve setting 2 The manifold is exposed to air Bio Dot Use for gravity filtration procedures Vacuum Air Flow valve setting 3 The manifold is exposed to both air and the vacuum Use this setting for gentle vacuum applications where the amount of vacuum is regulated by putting a finger over the port exposed to the air Bio Dot Vacuum Fig 3 Optional settings for the 3 way flow valve to obtain optimal performance from the Bio Dot apparatus 3 2 Helpful Hints 1 During the assay do not leave the vacuum on This may dehydrate the membrane and may cause halos around the wells Apply vacuum only until solutions are removed from the sample wells then adjust the flow valve so that the unit is exposed to a
16. d approximately 2 3 feet below the level of the apparatus usually to a waste receptacle on the floor With this increased hydrostatic pressure fluid will drain from the apparatus in 3 4 minutes This type of gentle pressure is also useful for binding nucleic acids to nitrocellulose or Zeta Probe membranes Section 4 Protein Blotting 4 1 Immunoassay Procedure Detailed instructions including a comprehensive troubleshooting guide for performing immunoassays are included in the Immun Blot instruction manuals 1 Assemble the Bio Dot apparatus as described in Section 3 Prewet the membrane prior to placing it in the apparatus Nitrocellulose membranes are prewetted in TBS nylon membranes such as the Zeta Probe membrane are prewetted in distilled water see Section 9 for solution preparation Make sure that all the screws have been tightened under vacuum to ensure that there will not be any cross well contamination Notes Zeta Probe membranes must be removed from the Bio Dot apparatus after the antigen is immobilized The blocking and other incubation steps should be carried out ina separate container Zeta Probe membranes require more stringent blocking conditions using 5 w v nonfat milk or 3 w v gelatin in 1x TBS which cannot be filtered through the membrane using the Bio Dot apparatus Rehydrate the membrane to ensure uniform binding of the antigen Use 100 ui TBS per well for nitrocellulose membranes Use 100 u distilled
17. e can be dried When reprobing do not allow the membrane to dry between hybridizations Expose moist membranes between plastic wrap or enclosed in a heat sealable plastic bag Do not allow a wet membrane to come in contact with the film because a wet Zeta Probe membrane will stick to the film and moisture on the film can cause artifacts Note To increase the rate of hybridization include 10 dextran sulfate final concentration in the hybridization solution Maniatis et al 1982 Prewarm hybridization solution to 50 C Denature the probe and carrier as above Special care must be taken to ensure uniform mixing of the denatured probe with the hybridization solution since the solution is quite viscous at 50 C 7 4 Probe Stripping and Rehybridization If reprobing is desired do not allow the membrane to dry between hybridizations The membrane should be stripped as soon as possible after autoradiography 1 Wash 2 times 20 minutes each in a large volume of 0 1x SSC 0 5 SDS at 95 C 2 Check membrane for removal of autoradiography patterns by overnight exposure 14 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Section 8 Solutions for Protein Applications 8 1 Solutions for Nitrocellulose Membrane Tris Buffered Saline 1x TBS 2 L 20 mM Tris HCl pH 7 5 500 mM NaCl Dissolve 4 84 g Tris 58 48 g NaCl in 1 5 L ddH O Adjust to pH 7 5 with HCI Adjust the volume to
18. e not interchangeable If Method A is chosen all solutions must be preparied according to Method A if Method B is chosen all solutions must be prepared according to Method B TBS Tris Buffered Saline 2 L Same as nitrocellulose membrane solution 15 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com TTBS Tween 20 Tris Buffered Saline 2 L Method A Add 3 ml Tween 20 to 1 L of TBS This solution is used when nonfat dry milk is the blocking agent OR Method B Add 2 ml Tween 20 and 50 ml MPO to 1 L of TBS This solution is used when gelatin and MPO are the blocking agents Blocking Solution 100 ml Method A Add 5 g of nonfat dry milk to 100 ml of TBS OR Method B Add 3 g of gelatin to 100 ml of TBS Warm to 37 C to dissolve the gelatin with stirring Cool before use Antibody Buffer 200 ml Method A Add 10 g of nonfat dry milk to 200 ml TTBS 100 ml is used for the primary antibody solution and 100 ml is used for conjugate dilution OR Method B Add 2 g gelatin to 200 ml TTBS that already contains 5 MPO Warm to 37 TC to dissolve gelatin and cool before adding antibody 100 ml is used for the primary antibody solution and 100 ml is used for conjugate dilution Primary Antibody Solution 100 ml Same as nitrocellulose membrane antibody buffer solution Secondary Antibody Solution 100 ml Same as nitrocellulose membrane antibody buffer solution Color Develo
19. eled Probes 1 DNA and RNA a Unincorporated label small radioactive decay products and small probe fragments resulting from nick translation can increase overall background Use Bio Spin chromatography columns to remove unincorporated label Filter hybridization solutions before use Use the probe as soon as possible after preparation Reduce exposure of the probe to DNase during nick translation Improper blocking conditions were used Increase the blocker concentration Use a different heterologous nucleic acid in the prehybridization mixtures Sonicate the solution thoroughly and denature before use The blocker shares common sequences with host or vector of cloned probe Vary the blocker Yeast tRNA may be useful instead of salmon sperm DNA Cut the probe out of the vector and purify Washes were insufficient Include stringent washes i e increase the temperature of the washes or decrease the salt concentration Increase the number and the length of the standard washes The probe was too heavily labeled or concentrated Dilute the probe The incubation period was too long Shorten the reaction time The bag used in hybridization collapsed on the membrane Be sure the membrane is floating freely in the hybridization bag and that the volume of solution present is enough to prevent the bag from collapsing during incubations Dust was present on the membrane Remove by washing in 2x Denhardt s prior to baking or with a brief wa
20. eta Probe membrane denature the DNA sample by addition of NaOH and EDTA solution to final concentrations of 0 4 M NaOH 10 mM EDTA Heat the sample to 100 C for 10 minutes to ensure complete denaturation When applying DNA to nitrocellulose membrane denature the DNA in the same manner The DNA must then be neutralized by adding an equal volume of cold 2 M ammonium acetate pH 7 0 to the target DNA solution 2 Prewet the membrane by placing the membrane gently at a 45 angle into a tray of the wetting solution Always wear gloves when handling blotting membranes Nitrocellulose membranes should be wetted in 6x SSC Zeta Probe membranes should be wetted in distilled water see Section 10 for formulations 3 Assemble the Bio Dot apparatus according to the instructions in Section 3 1 Apply the vacuum and then retighten the screws that hold the apparatus together Rehydrate the membrane with 500 ul Tris EDTA TE or H O as described in Section 3 1 At this point the unit is ready for sample application 4 Samples and wash solutions should be applied with a standard pipet or a Costar Octapette pipet with the vacuum off and the flow valve open Apply the denatured DNA in a 50 500 ul sample volume Multiple loadings may be performed However best binding and most rapid results occur using minimum sample volumes Fill all wells with the same volume to obtain homogeneous filtration 5 The sample may be pulled through by applying a gentle vacuum
21. g membranes Prewet the blotting membrane by placing it gently at a 45 angle into a tray of wetting solution Wet the Zeta Probe membrane in distilled water nitrocellulose in 6x SSC see Section 9 for solution preparation Assemble the Bio Dot apparatus according to the instructions in Section 3 1 Remember to apply the vacuum and then retighten the screws that hold the apparatus together Immediately before use dissolve RNA samples in 500 ul of ice cold 10 mM NaOH 1 mM EDTA Samples and wash solutions may be applied with a standard pipet or a Costar Octapette pipet Apply the denatured RNA and pull the sample through by passive filtration or by applying a gentle vacuum Note A method for applying gentle vacuum to the apparatus is to adjust the flow valve to setting 3 Use a finger to cover the valve port exposed to air The amount of vacuum reaching the manifold will be regulated by the pressure of your finger on the valve Rinse all wells to wash through any sample on the side of the wells Rinse with 500 ul cold 10 mM NaOH 1 mM EDTA Apply vacuum flow valve setting 1 Figure 3 until the sample wells are dry Disassemble the Bio Dot apparatus Remove the blotted membrane and rinse it in 2x SSC 0 1 sodium dodecyl sulfate SDS Nitrocellulose membranes must be baked under vacuum for 2 hours at 80 before hybridization The Zeta Probe membrane is ready for hybridization If hybridization is not to be undertaken within 2 d
22. holes in the support plate Visually inspect the gasket to make sure the holes are properly aligned If the gasket is not centered pull lightly at the corners until it is aligned Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Sample template with attached sealing screws Sealing gasket Gasket support plate Vacuum manifold Fig 1 Diagram of proper Bio Dot apparatus assembly 4 Always use forceps or wear gloves when handling membranes Prewet the nitrocellulose or Zeta Probe membrane by slowly sliding it at a 45 angle into wetting solution Note PVDF membrane is not recommended Wet nitrocellulose in 6x salt sodium citrate SSC for nucleic acid applications and in Tris buffered saline TBS for protein blotting Wet Zeta Probe membrane in distilled water See Sections 8 and 9 for solution preparation A 10 minute soak is recommended for complete wetting of the membrane to ensure proper drainage of solutions Remove the membrane from the wetting solution Let the excess liquid drain from the membrane Touching the membrane to a sheet of filter paper is a simple method for removing excess buffer Lay the membrane on the gasket in the apparatus so that it covers all of the holes The membrane should not extend beyond the edge of the gasket after the Bio Dot apparatus is assembled Remove any air bubbles trapped between the membrane and the gasket Note PVDF membrane
23. io Rad s GS 800 densitometer 2 Assay for Particular Antigen or Target Cell Antigen a Place a prewetted filter paper Whatman GF B in the Bio Dot apparatus Attach the sample template and tighten the screws Fill all the wells with buffer and apply a vacuum With the vacuum on and the buffer draining retighten the screws The presence of buffer while applying vacuum will help prevent the filter paper from breaking When the buffer is gone turn off the vacuum and close the flow valve b Add 50 Wl fetal bovine serum FBS 10 v v in blocking buffer Allow the FBS buffer to incubate for 10 minutes then open the flow valve and filter through by gravity c Add approximately 12 500 target cells in 50 ul FBS buffer to each well Gently pull the solution through the membrane by attaching tubing to the flow valve to increase the hydrostatic pressure see Section 3 2 Perform 3 washes with TBS buffer using tubing rather than vacuum to speed the flow rate d Perform antibody incubations as described in Section 4 1 for protein immunoassays Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Section 5 DNA Blotting This section gives protocols for DNA blotting Both the alkaline blotting method using Zeta Probe membrane and the standard method for DNA blotting to nitrocellulose are described 1 The target DNA must be denatured prior to application to the membrane When using the Z
24. ion 1 SDS 0 1 SDS 0 5 nonfat milk 100 pg ml denatured salmon sperm DNA Section 10 Troubleshooting Guide l Filtration Apparatus 1 2 Leakage or Cross Well Contamination a b Improper assembly The screws must be retightened under vacuum following the initial assembly Membrane is not properly rehydrated after assembly Always rehydrate the membrane prior to applying samples Apply vacuum only until solutions are removed from the sample wells then disconnect the vacuum source No Filtration or Uneven Filtration Occurring a Macromolecular polymers cellular debris or dirt is plugging the membrane Centrifuge samples prior to application to remove particulates Filter solution prior to use to ensure removal of particulate material Cover wells with Parafilm during lengthy incubations Bubbles are obstructing the filtration Use a needle to break any bubbles being careful not to puncture the membrane Pipet liquid in the wells up and down to displace bubbles The flow valve is positioned higher than the apparatus The flow valve must be lower than the level of the sample wells on the apparatus for proper drainage to occur Improper blocking reagent is used BSA is the recommended blocker for nitrocellulose gelatin will plug the apparatus and no filtration will occur The Zeta Probe membrane which requires more stringent blocking with nonfat milk or with gelatin and MPO should be removed from the Bio Dot a
25. ion 10 Section 11 Section 12 12 1 12 2 Section 13 Table of Contents Page DVER COUN CEN n Ea E E E A 1 SpecMiCatlONS ainsa e A a P EEEE EER 1 Special Handling Features cccccsssecseseeceeesseeeeeeeseeeeeenseeeees 1 AUTO CIAVING sist ceeds cacescecscpeetarssagevaace te a a aada 1 Chemical Stability eiei ineei nine 2 Blo Dat Assembly xicesseesnce neice eicecd eeccsis dicen etnies 2 PASS SIMD V5 ccs wer E ch tate E E E 2 Fel OU WINS c E 5 Protein BIOTIC ssiiscsist ina cterdatencnceessininecsniedsteetcinisbeeseatendcantienasne 6 Immunoassay Procedure eee eeeeeeceeeeenneeeeeeeaeeeeeeeeaaeeeeeeeaeeeeeeeaaeeeeeeenaes 6 Special Protein Blot Applications c ccccccceeeceeseeeeeeeeeeeeeaeeeeeeeeessaeetennees 8 DNA Blotting ss sissies cseses cansensectesansteenssussasenarasc eentsaeaneneansancaneeens 9 RNA BO CU Gs ssscsscccs ci ctctcrssatnencte ces stnerecntesaaeni sence sacasnnneeanarersenienunanain 9 Alkaline RNA Denaturation and Fixation 0 cccesceeeeeeeeeeeeeeseteeeeeeeees 10 Glyoxal RNA Denaturation and Fixation 0 cccccceeeseeeseereeeeteeeeeeeees 10 Hybridization Protocols for Nucleic ACIS ss eeeeeeeeeenees 11 Probe Recommendations cece eeeceee sent ee eee eete eee ee taaeeeeeetaeeeeeetaaeeeene 11 Hybridization Protocols for DNA or RNA Bound to Nitrocellulose or Zeta Probe MEMbrAane eeeeccceeeeseeeceeeeenneeeeeeeaeeeeeeeaaaeeeeeeeaaeeeeeeeaaas 12 Hybridizati
26. ion 13 Ordering Information Catalog Description 170 6545 Bio Dot Microfiltration Apparatus includes Bio Dot sample template vacuum manifold base plate membrane support and gasket 170 6547 Bio Dot Module includes Bio Dot sample template membrane support and gasket 170 6546 Bio Dot Gaskets 3 gaskets per package 170 6542 Bio Dot SF Microfiltration Apparatus includes Bio Dot SF sample template vacuum manifold base plate membrane support gasket and filter paper 170 6543 Bio Dot SF Module includes Bio Dot SF sample template membrane support gasket one conversion of the Bio Dot apparatus to the Bio Dot SF apparatus 170 6544 Bio Dot SF Gaskets 2 gaskets per package 162 0161 Bio Dot SF Filter Paper 60 sheets 162 0117 Nitrocellulose Membrane 0 45 um for Bio Dot SF applications 9 x 12 cm sheets 10 162 0153 Zeta Probe Membrane for use with the Bio Dot apparatus and Bio Dot SF apparatus 9 x 12 cm sheets 15 732 6000 Bio Spin 6 columns 732 6004 Bio Spin 30 columns 142 6425 AG 501 X8 D resin H OH 20 50 mesh 500 g 24 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com foR Ve Bio Rad Laboratories Inc Life Science Web site www bio rad com USA 800 4BIORAD Australia 02 9914 2800 Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 21 2527 3454 Canada 905 712 2771 China 86 21 63052255 Czech Republic 420 2 41 43 05 32 Denmark 44 52 10 00 Finla
27. ir and disconnect the vacuum If some sample wells are not used in a particular assay those wells must be closed off to ensure proper vacuum to the wells in use There are three ways to close off unused wells One is to apply a 3 gelatin solution to those wells Gelatin will clog the membrane and cut off the vacuum flow to the clogged wells The second method is to cover the unused portion of the apparatus with tape to prevent air from moving through those wells The third method is to add buffer to the empty wells at each step instead of sample or wash solutions If an overnight incubation is desired adjust the flow valve so that the vacuum manifold is closed off from both the vacuum and air before applying samples see Figure 3 In this configuration solutions will remain in the sample wells with less than 10 loss of volume during an overnight incubation Note that the unit must be kept at a constant temperature during extended incubations If the unit cools more than 10 C 20 F a partial vacuum will build inside the unit and drainage will occur Any particulate in samples or solutions will block the membrane and restrict flow of solutions through the membrane For best results filter or centrifuge samples to remove particulate matter Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Check the wells after sample has been applied to ensure that there are no air bubbles in the wel
28. is not recommended Place the sample template on top of the membrane The guide pins ensure that the template will be properly aligned Finger tighten the four screws When tightening the screws use a diagonal crossing pattern to ensure uniform application of pressure on the membrane surface see Figure 2 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com J Outlet port Fig 2 Diagonal crossing pattern for tightening screws in the Bio Dot apparatus 6 Attach a vacuum source house vacuum or a vacuum pump to the flow valve with a waste trap set up and positioned between the vacuum outlet and the flow valve Turn on the vacuum and set the 3 way valve to apply vacuum to the apparatus flow valve setting 1 Figure 3 7 With vacuum applied repeat the tightening process using the diagonal crossing pattern Tightening while vacuum is applied ensures a tight seal preventing cross contamination between slots Failure to tighten screws during application of vacuum prior to starting the assay may lead to leaking between the wells 8 Adjust the flow valve so that the vacuum manifold is open to the air flow valve setting 2 Figure 3 Apply 100 pl buffer to all 96 sample wells Use of the 8 channel pipet and buffer reservoirs see Section 13 for ordering information will simplify the process of adding solutions to the Bio Dot apparatus Addition of buffer is necessary to rehydrate the mem
29. lity The Bio Dot apparatus can be used with 100 alcohol solutions and concentrated alkali or acid solutions It cannot be used with aromatic or chlorinated hydrocarbons see Table 1 Section 2 Special Handling Features The Bio Dot apparatus withstands autoclave temperatures for sterilization as well as cleaning with alcohols acids and basic solutions 2 1 Autoclaving The Tygon tubing and flow valve cannot be autoclaved All other components of the apparatus withstand the autoclave treatment After repeated autoclaving 25 cycles the silicone rubber gasket may need replacing The autoclave conditions that should be used are a maximum sterilization temperature of 250 F 121 C for 15 minutes followed by a 1 minute fast exhaust Higher temperatures or increased exposure times will significantly Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com reduce the life of the apparatus Do not autoclave the unit with the thumbscrews tightened as this may cause the unit to warp during exposure to the elevated temperatures 2 2 Chemical Stability The apparatus is stable in acid and base solutions as well as alcohol solutions This feature allows rapid cleanup and sterilization of the apparatus and gaskets The unit is not compatible with polar aromatic or chlorinated hydrocarbons esters and ketones These solvents will cause degradation of the plastic See Table 1 for list of chemical
30. llulose or Zeta Probe Membrane Prehybridization 1 Place the blotted membrane inside a heat sealable plastic bag Seal three sides leaving the top side open 2 Pipet in the correct prehybridization solution for application For DNA or RNA Bound to For DNA Bound to For RNA Bound to Zeta Probe Membrane Nitrocellulose Nitrocellulose See instruction manual Maniatis et al 1982 Thomas 1980 1 mM EDTA 6x SSC 50 formamide 7 SDS 0 5 SDS 5x SSC 0 5 M NaHPO pH 7 2 5x Denhardt s solution 1x Denhardt s solution 100 ug ml denatured 50 mM NaHPO pH 6 5 salmon sperm DNA 250 ug ml denatured 1 mM EDTA salmon sperm DNA The carrier DNA used with nitrocellulose must be denatured before adding it to the prehybridization solution Heat the DNA at 100 C for 5 minutes and cool rapidly 3 Seal the top of the bag and incubate For DNA or RNA Bound to For DNA Bound to For RNA Bound to Zeta Probe Membrane Nitrocellulose Nitrocellulose 5 minutes at 65 C 2 4 hours at 68 C 8 20 hours at 42 C Hybridization 1 Cut one corner of the plastic bag Remove the prehybridization solution and replace it with a fresh batch of the same solution except when binding RNA to nitrocellulose In that case add 10 dextran sulfate to the hybridization solution Note formamide can also be used in the hybridization buffer to lower the incubation temperature when binding DNA to nitrocellulose or Zeta Probe membrane Maniatis et al 1982 Casey and Davidson
31. ls Air bubbles will prevent the sample from binding to the membrane Air bubbles may be removed by pipetting the liquid in the well up and down Proper positioning of the flow valve relative to the level of the apparatus is important for proper drainage The speed of filtration is determined by the difference in hydrostatic pressure between the fluid in the sample wells and the opening of the flow valve which is exposed to air If the opening of the flow valve is above the level of the sample wells very little drainage will occur When the flow valve is positioned at a level below the sample wells proper drainage will occur during filtration applications The best method for removing the blotted membrane from the Bio Dot apparatus is to leave the vacuum on following the wash step With the vacuum on loosen the screws and remove the sample template Next turn off the vacuum and remove the membrane A method for applying gentle vacuum to the apparatus is to adjust the flow valve so that it is open to air the vacuum source and the vacuum manifold while the vacuum is on Then use a finger to cover the valve port exposed to the air The amount of vacuum reaching the manifold will be regulated by the pressure of your finger on the valve For applications using glass membranes that might break under vacuum pressure an extra piece of tubing can be attached to the flow valve to increase hydrostatic pressure during wash steps This tubing should exten
32. ncentration of the solutes and enhancing hybridization Exposure time was insufficient Increase the time of exposure Sample load was insufficient Increase the sample load Probe concentration is too low If low signal is accompanied by low background then the probe concentration can be increased Binding of nucleic acid to the membrane was incomplete See Troubleshooting Part Il If no autoradiographic signal is seen make sure the probe was denatured by heating to 100 C by exposure to 0 4 N NaOH or by heating to 65 C for 5 minutes in 50 formamide prior to hybridization 2 Protein d e f Antigen binding was incomplete See Troubleshooting Part Il Monoclonal antibodies may not recognize a denatured antigen Asses the binding of other monoclonal or polyclonal antibodies Blot only native proteins The enzyme conjugate or the substrate has been inactivated Primary or secondary antibody is inactive or nonsaturating Test the enzyme antibody and substrate separately for activity Increase concentration of the primary or secondary antibody Eliminate detergents from reactions and washes With HRP avoid sodium azide as it is a potent inhibitor of the enzyme For labeled probes exposure time was insufficient Increase the time of exposure Antibody reaction times are insufficient Increase reaction times Sample load was insufficient Increase the concentration of antigen applied V Nonspecific or Nonquantitative De
33. nd 09 804 22 00 Gr oup France 01 47 95 69 65 Germany 089 318 84 0 Hong Kong 852 2789 3300 Hungary 36 1 455 8800 India 91 124 6398112 113 114 6450092 93 Israel 03 951 4127 Italy 39 02 216091 Japan 03 5811 6270 Korea 82 2 3473 4460 Latin America 305 894 5950 Mexico 55 52 00 05 20 The Netherlands 0318 540666 New Zealand 64 9 415 2280 Norway 23 38 41 30 Poland 48 22 331 99 99 Portugal 351 21 472 7700 Russia 7 095 721 1404 Singapore 65 6415 3188 South Africa 00 27 11 4428508 Spain 34 91 590 5200 Sweden 08 555 12700 Switzerland 061 717 9555 Taiwan 8862 2578 7189 2578 7241 United Kingdom 020 8328 2000 Sig 1103 M1706545 Rev C Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com A rtisan Artisan Technology Group is your source for quality TecmoogyGroup new and certified used pre owned equipment FAST SHIPPING AND SERVICE CENTER REPAIRS WE BUY USED EQUIPMENT DELIVERY Experienced engineers and technicians on staff Sell your excess underutilized and idle used equipment TENS OF THOUSANDS OF at our full service in house repair center We also offer credit for buy backs and trade ins IN STOCK ITEMS www artisantg com WeBuyEquipment 7 EQUIPMENT DEMOS HUNDREDS OF InstraV ea REMOTE INSPECTION LOOKING FOR MORE INFORMATION MANUFACTURERS Remotely inspect equipment before purchasing with Visit us on the web at www artisantg com 7 for more our interactive website at www instraview com inf
34. ne receptor assays Gershoni and Palade 1983 Schafer et al 1974 Immunoglobulin detection Herbrink et al 1982 Wang et al 1980 Hybridoma screening Bennett and Yeoman 1983 Hawkes et al 1982 Horejsi and Hilgert 1983 Kane et al 1982 Locker and Motta 1983 Shen et al 1980 DNA binding proteins Achberger and Whiteley 1981 Allen and Parsons 1979 Karagyozov and Hadjiolov 1982 Lin and Riggs 1975 Lye and Birge 1981 Glycoprotein lectin assays Gershoni and Palade 1983 Neuhoff et al 1981 Viral antigen analysis clinical applications Cleveland et al 1981 Richman et al 1981 1982 Column or gel column fraction monitoring Cunningham 1983 Palfree and Elliott 1982 Shen et al 1980 Antibody purification Olmsted 1981 Total protein microassay Nakamura et al 1985 21 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Nucleic Acid Dot hybridization of DNA RNA for sequence homology sequence abundance etc Kaftos et al 1979 Thomas 1980 Antibody detection of DNA Tron et al 1983 e Viral DNA sequence detection Berg et al 1986 Brandsma and Miller 1980 e Hybridization selection Harpold et al 1978 Ricciardi et al 1979 Gene product and clone selection Kranz and Gennis 1982 e Plasmid analysis Bresser and Gillespie 1983 e CsCl gel column or sucrose gradient fraction Cunningham 1983 Palfree and Elliott 1982 Shen et al 1980 e DNA fil
35. ng do not allow the membrane to dry between hybridizations Make the autoradiographic exposure with the moist membrane wrapped in plastic wrap Do not allow the wet membrane to come in contact with the film because a wet Zeta Probe membrane will stick to the film and any moisture on the film will cause artifacts black spots 7 3 Hybridization Protocols for RNA Probes The following protocols are for RNA probes to DNA blots Casey and Davidson 1977 contains protocols for RNA RNA hybridizations Prehybridization 1 Place the blotted membrane inside a heat sealable plastic bag Seal three sides leaving the top side open Pipet in the prehybridization solution For DNA Bound to For DNA Bound to Zeta Probe Membrane Nitrocellulose See instruction manual Jerome and Jaehning 1986 50 formamide 50 formamide 1 5x sodium sodium phosphate EDTA SSPE 0 1 SDS 1 SDS 5x SSPE 0 5 nonfat dry milk 5x Denhardt s solution 200 ug ml carrier RNA 200 ug ml denatured salmon sperm DNA 500 ug ml denatured salmon sperm DNA The DNA must be denatured before adding it to the prehybridization solution by heating at 100 C for 5 minutes followed by rapid cooling in ice 3 Seal the bag and incubate DNA Bound to DNA Bound to Zeta Probe Membrane Nitrocellulose 30 minutes at 50 C 4 hours at 42 C 13 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Hybridization 1
36. oassay using 5l labeled staphylococcal protein A for anti body to varicella zoster virus J Infect Dis 143 693 699 1981 Richman DD et al A rapid enzyme immunofiltration technique using monoclonal antibodies to serotype herpes simplex virus J Med Virol 9 299 305 1982 Schafer A et al A solid phase radioimmunoassay for urine aldosterone using antibodies linked to nylon nets FEBS Lett 48 230 234 1974 23 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Shen V et al Monoclonal antibodies to Escherichia coli 50S ribosomes Nucleic Acids Res 8 4639 4649 1980 Smith LH et al Human monoclonal antibody recognizing an antigen associated with ovarian and other adenocarcinomas Am J Obstet Gynecol 166 634 645 1992 Thomas P Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose Proc Natl Acad Sci USA 77 5201 5205 1980 Tron F et al Clinical and theoretical interest of the immunochemical analysis of antinuclear antibod ies in systemic lupus erythematosus Adv Nephrol Necker Hosp 12 215 237 1983 Wang R et al A simplified solid phase immunofluorescence assay for measurement of serum immunoglobulins Clin Chim Acta 102 169 177 1980 Winberg G and Hammarskjold ML Isolation of DNA from agarose gels using DEAE paper Application to restriction site mapping of adenovirus type 16 DNA Nucleic Acids Res 8 253 264 1980 Sect
37. on Gently agitate the solution until development is complete then remove the membrane and rinse it in distilled water to stop the reaction Place the membrane on filter paper to air dry 13 When using HRP color development substrate wash each well twice with 200 ul TBS to eliminate excess Tween 20 This wash step is not necessary when using AP systems or NBT BCIP color development Add 100 200 ul of the color development solution to each well The reagent can be allowed to react while the solution slowly drains by gravity filtration or the reaction time can be extended by closing the flow valve prior to adding the substrate In either application when the color development is completed the excess substrate should be removed by vacuum and all the sample wells should be vacuum washed with 200 ul of distilled water to stop the reaction Following this wash step remove the membrane from the apparatus Rinse the membrane in distilled water and allow it to airdry on filter paper 4 2 Special Protein Blot Applications 1 Soluble Enzyme Substrate Reactions and Quantitations Perform an immunoassay as described in Section 4 1 Prior to color development disconnect the vacuum and close the flow valve Add an equal volume of substrate solution to all wells Visualize positive reactions and record For quantitation withdraw equal aliquots of the soluble substrate reactant from each well and transfer to a disposable plastic microplate Quantitate using B
38. on Protocols for RNA Probes ccsecceeeseeeeeeeeeeseteeeeeeeeeees 13 Probe Stripping and Rehybridization cccceccececeeeeeeeeeeeeeeeeeeeseeeeenees 14 Solutions for Protein Applications cccsseecseseeeeeseeeeeeeeees 15 Solutions for Nitrocellulose Membrane ccccceceeeeeeteeeeeeeteeeeeeeees 15 Solutions for Zeta Probe Membrane for Protein Applications 15 Solutions for Nucleic Acid Applications cccessseeeeeeee 16 Troubleshooting Guide ccssseeeeceseeeeeeeeseeeeeeesseeeeeesseeeeeeesees 18 Legal NOU COS ise oe ciessa ces cetera rcesasscnceantansececeacia aeeee neater tees 21 Applications and References ccccesseseeeeecereeenseeeeeeneneeees 21 COMMON applications ccceceeceeeeee cece ee eeceeeeeeeeseaeeeeeaaeeseeeeeetiaeeeteneees 21 Referente Sicana sianidi aed ii eE E sve derntel auvanued EANN 22 Ordering Intorimatln sssisiciiesceieeessseticancanecenasseceananncsawcenaantenmencewens 24 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Section 1 Introduction The Bio Dot microfiltration apparatus can be used for any application requiring rapid immobilization and screening of unfractionated or purified proteins nucleic acids or macromolecular complexes on membranes such as nitrocellulose or Zeta Probe membrane The Bio Dot apparatus is provided as a complete unit or as a modular
39. on the valves 6 Rinse all wells to wash through any sample on the side of the wells Rinse with 500 ul TE Apply vacuum flow valve setting 1 Figure 3 until the sample wells are dry 7 Disassemble the Bio Dot apparatus Remove the blotted membrane 8 Remove glyoxal adducts by pouring 20 mM Tris HCl pH 8 0 1 mM EDTA heated to 95 C onto the membrane and agitating at room temperature until the solution cools Place the membrane in a vacuum oven at 80 C for 1 hour for the Zeta Probe membrane 2 hours for nitrocellulose 9 Nitrocellulose membrane must be baked under vacuum for 2 hours at 80 C before hybridization If hybridization is not to be undertaken within 2 days then bake the Zeta Probe membrane under vacuum for 30 minutes at 80 C Zeta Probe and nitrocellulose membranes can be stored dry between two pieces of filter paper in plastic bags at 23 25 C Section 7 Hybridization Protocols for Nucleic Acids 7 1 Probe Recommendations The specific activity concentration size range and purity of the probe all have an important effect on signal to noise ratio during hybridization For hybridization on Zeta Probe membrane the following is recommended Probe specific activity 108 com ug probe Probe concentration in the hybridization mixture 108 counts ml 10 50 mg ml Probe length 200 1 000 bp Optimal probe specific activity and concentration can vary according to available hybridization sites and exposure time Alterna
40. ormation on price quotations drivers technical LEASING MONTHLY specifications manuals and documentation RENTALS ITAR CERTIFIED D a gaa tia Contact us 888 88 SOURCE sales artisantg com www artisantg com
41. pment Solution The specific chemicals and buffers are dependent on the enzyme conjugate being used See the Bio Rad Immun Blot assay kit instruction manual for details on how to make the appropriate solution Section 9 Solutions for Nucleic Acid Applications 20x SSC 3 M NaCl 0 3 M trisodium citrate FW 294 1 Dissolve 175 0 g NaCl and 88 2 g trisodium citrate in ddH O Adjust volume to 1 L with ddH O 20x SSPE 3 6 M NaCl 0 2 M Na HPO 7H O 0 02 M EDTA Dissolve 210 0 g NaCl 53 6 g Na HPO 7H O 7 44 g EDTA ddH O Adjust volume to 1 L with ddH O 16 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com TE 10 mM Tris HCl pH 8 0 1 mM EDTA pH 8 0 Dilute 10 ml 1 M Tris HCl pH 8 0 and 4 ml 0 250 M EDTA pH 8 0 to 1 L with ddH O 100x Denhardt s Solution 2 bovine serum albumin 2 polyvinylpyrrolidone 2 Ficoll Dissolve 2 0 g BSA 2 0 g polyvinylpyrrolidone 2 0 g Ficoll in ddH O Adjust volume to 100 ml with ddH O 20 SDS Dissolve 200 0 g SDS in ddH O Adjust volume to 100 ml with ddH O It may be necessary to heat to 65 C to get into solution 1 M NaHPO pH 7 2 Dissolve 134 0 g Na HPO 7H O F W 268 07 in ddH O Add 4 ml 85 H PO Adjust volume to 1 L with ddH O 50 Dextran Sulfate 50 dextran sulfate 0 2 sodium azide Dissolve 50 0 g dextran sulfate and 0 2 g sodium azide in ddH O Adjust volume to 100 ml with ddH O Store at 4 C 50
42. pparatus following antigen immobilization and the rest of the assay should be conducted in a separate container 3 Halos a Membrane is not properly rehydrated before applying samples Always rehydrate membrane prior to applying any sample Excessive concentrations of sample are loaded When too much sample is present wicking into the membrane around the well will occur Use serial dilutions of the samples to determine optimal amounts to load ll Poor Binding to Membrane 1 Nitrocellulose a DNA RNA will only bind efficiently in 20x SSC or 1 M ammonium acetate Use Zeta Probe membrane as an alternative 18 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com b DNA must be single stranded and RNA must be denatured DNA lt 500 bp may not bind to nitrocellulose Use Zeta Probe membrane as an alternative Mixed ester cellulose binds DNA RNA and protein very poorly Use Bio Rad s pure nitrocellulose Proteins lt 15 000 daltons may show decreased binding to 0 45 um nitrocellulose Use Zeta Probe membrane or 0 2 um nitrocellulose Also glutaraldehyde fixation will increase retention of small proteins and peptides to both nitrocellulose and Zeta Probe membrane Protein may be removed from nitrocellulose by SDS NP 40 or Triton X 100 Use Tween 20 in washes Reduce concentrations or time of any SDS or NP 40 washes lll High Background After Incubation With Lab
43. ridoma cultures J Immunol Methods 62 325 329 1983 Huet J et al Spot immunodetection of conserved determinants in eukaryotic RNA polymerases Study with antibodies to yeast RNA polymerases subunits J Biol Chem 257 2613 2618 1982 Jantzen K et al The DNase sensitive domain of the chicken lysozyme gene spans 24 kb Nucleic Acids Res 14 6085 6099 1986 Jerome JF and Jaehning JA mRNA transcription in nuclei isolated from Saccharomyces cerevisiae Mol Cell Biol 6 1633 1639 1986 Johnson DA et al Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose Gene Anal Tech 1 3 8 1984 Kaftos FC et al Determination of nucleic acid sequence homologies and relative concentrations by a dot hybridization procedure Nucleic Acids Res 7 1541 1552 1979 Kane CM et al Tissue specific and species specific monoclonal antibodies to avian red cell nuclear proteins Proc Natl Acad Sci USA 79 6265 6269 1982 Karagyozov LK and Hadjiolov AA Isolation of active transcription complexes from animal cell nuclei by nitrocellulose binding J Biochem Biophys Methods 5 329 339 1982 Kranz RG and Gennis RB A quantitative radioimmunological screening method for specific gene products Anal Biochem 127 247 257 1982 Kutateladze TV et al New procedure of high voltage electrophoresis in polyacrylamide gel and its application to the sequencing of nucleic acids Anal Biochem 100 12
44. sh prior to hybridization The gasket is contaminated by radioactivity Replace the gasket 2 Protein a Impure secondary antibody was used Use Bio Rad s affinity purified blotting grade second antibody Excessive reaction time in the substrate Remove the blot from the substrate reaction when the signal to noise level is acceptable Improper blocking conditions were used Be sure the blocker is pure protein Increase the blocker concentration or blocking time Match the blocker with the detection system Hemoglobin reacts with HRP BSA may contain IgG contaminants 19 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com d e Primary or secondary antibody is too concentrated Dilute the antibodies Washes were insufficient Increase the number and or duration of the washes Include progressively stronger detergents in the washes For example SDS gt NP 40 gt Tween 20 Also include Tween 20 in the antibody buffers to reduce nonspecific binding IV Poor Detection Sensitivity or No Reactivity 1 DNA RNA a This problem may occur when total genomic DNA is probed for single copy or low copy number genes Try the Zeta Probe membrane for binding and retention of increased quantities of DNA Hybridization was insufficient Incorporate 10 dextran sulfate in the hybridization mixture This polymer effectively reduces the solvent volume thereby increasing the co
45. tection 1 Protein a b Monoclonal antibodies may react nonspecifically with SDS denatured proteins Compare binding of other monoclonal or polyclonal antibodies Blot native proteins Concentration of the primary or secondary antibody is excessive Increase the dilution of the antibodies 20 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com c Primary or secondary antibody is contaminated with nonspecific or species cross reactive IgG Use a purified IgG primary antibody fraction and affinity purified blotting grade secondary antibody d Slow gentle filtration is needed for complete optimal protein binding 2 DNA RNA a Probe is not pure b Blocker shares common sequences with the probe Assess different blockers Use more stringent washes Section 11 Legal Notices Ficoll is a trademark of Amersham Biosciences Parafilm is a trademark of American National Can Co Teflon and Tygon are trademarks of E I DuPont de Nemours amp Co Triton is a trademark of Union Carbide Chemicals amp Plastics Technology Corp Tween is a trademark of ICI Americas Inc Section 12 Applications and References 12 1 Common Applications Protein Radioimmunoassay RIA enzyme linked immunoassay EIA fluoroimmunoassay FIA of soluble or particulate cellular antigens Cleveland et al 1981 Shen et al 1980 Analysis of enzymes Faulstich et al 1974 Huet et al 1982 Hormo
46. ter binding assays including DNA drug DNA protein virus host DNA synthesis etc Bresser and Gillespie 1983 e DNA RNA purification Chen and Thomas 1980 Holland and Wangh 1983 Kutateladze et al 1979 Winberg and Hammarskjold 1980 12 2 References Achberger EC and Whiteley HR The role of the delta peptide of the Bacillus subtilis RNA polymerase in promoter selection J Biol Chem 256 7424 7432 1981 Allen JD and Parsons SM Nitrocellulose filter binding quantitation of the histidyl tRNA ATP phosphoribosyltransferase complex Anal Biochem 92 22 30 1979 Bennett FC and Yeoman LC An improved procedure for the dot immunobinding analysis of hybridoma supernatants J Immunol Methods 61 201 207 1983 Berg LJ et al Complementation of a bovine papilloma virus low copy number mutant evidence for a temporal requirement of the complementing gene Mol Cell Biol 6 859 869 1986 Bio Rad Laboratories Zeta Probe Membrane Instruction Manual Brandsma J and Miller G Nucleic acid spot hybridization rapid quantitative screening of lymphoid cell lines for Epstein Barr viral DNA Proc Natl Acad Sci USA 77 6851 6855 1980 Bresser J and Gillespie D Quantitative binding of covalently closed circular DNA to nitrocellulose in Nal Anal Biochem 129 357 364 1983 Casey J and Davidson N Rates of formation and thermal stabilities of RNA DNA and DNA DNA duplexes at high concentrations of formamide Nucleic Acids Res 4 1539 1552 197
47. tive hybridization protocols are necessary when probe lengths vary outside this recommended range See Zeta Probe membrane instruction manual Probe cleanup is essential to minimize background Unincorporated nucleotides present after probe preparation contribute to hybridization background The most effective cleanup method is by column chromatography This can be done quickly and easily with Bio Spin chromatography columns Bio Spin 6 columns catalog number 732 6000 or Bio Spin 30 columns catalog number 732 6004 After cleanup denature double stranded probes by heating to 95 100 C for 5 minutes Then cool rapidly on ice Use the probe as soon as possible after preparation Several hybridization protocols are given in this section All protocols are for using DNA probes to hybridize to either DNA or RNA The 7 SDS hybridization protocol requires minimal prehybridization treatment and has a high signal strength and low background Further references and techniques for hybridizing to the Zeta Probe membrane may be found in the Zeta Probe membrane instruction manual The final volume of hybridization solution is important in reducing background For prehybridization and hybridization use 150 ul solution cm of membrane For washes use at least 350 ul cm of membrane 11 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 7 2 Hybridization Protocols for DNA or RNA bound to Nitroce
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