User Manual - Galileo Bioscience
Contents
1. Trnovsky Jan Semi Dry Electroblotting of DNA and RNA from Agarose and Polyacrylamide Gels Biotechniques 13 800 804 1992 Blotting Hybridization amp Detection An S amp S Laboratory Manual a publication of Schleicher and Schuell 1995 This publication written by a leading manufacturer of blotting membranes gives protocols for the transfer of both proteins and nucleic acids Hybond Blotting Guide The direct route to excellent blotting results Amersham Life Science This publication gives very helpful hints and tips for producing good Western Northern and Southern blots along with a useful reference list Also included is a very useful troubleshooting guide for nucleic acid and protein blots with pictures of the problems descriptions of the symptoms and proposed solutions 13 TROUBLESHOOTING PROBLEM Transfer Efficiency is Poor Cause Current is too Low Solution Semi Dry Transfer should be performed at constant current Current density should be between 0 5 and 3mA cm of stack surface area Cause Power Supply is inappropriate for Semi Dry Transfer Solution Many power supplies will shut off or blow a fuse when run at the conditions required for semi dry transfer Semi dry transfer requires low voltage often less than 10V and high current Check with the manufacturer of the power supply to determine whether it is appropriate for semi dry transfer Cause Tr2. cm gel surface area 3mA cm gel surface area 10 14 Volts maximum 10 14 Volts maximum Running Time 30 minutes to 2 hours 30 minutes to 2 hours Needs to be experimentally generally in the lower range determined large molecules need longer transfer time TECHNICAL INFORMATIONAL TIPS Transfer Times Transfer times must be determined experimentally This is because transfer time is dependent upon Percentage of gel Type and amount of cross linking in the gel Type of protein cytoplasmic membrane nuclear Size of protein There is no formula for determining transfer time There are too many variables involved to give specific transfer conditions that will work for every protocol The guidelines of 2mA cm of gel for 1 hour are just a starting point exact conditions must be determined 11 12 References 1 Bjerrun O J and Schafer Nielsen C in Dunn J J ed Electrophoresis 86 VCH Weinheim 1986 pp 315 327 These authors compare results using different transfer buffers Towbin buffer vs the three buffer system Khyse Anderson J 1984 Electroblotting of multiple gels A simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide gels to nitrocellulose J Biochem Biophys Methods 10 203 209 This paper describes a semi dry blotter with a 3 Buffer system that is effective for the transfer of proteins Protein Blotting 3 Castora Frank J Western Blotting of Protein
3. Acetate 0 001M Disodium EDTA pH 8 0 1X Tris Glycine SDS Buffer TGS 1X TGS Buffer is used for denaturing polyacrylamide gel electrophoresis of proteins Final 1X Composition 0 025M Tris Base 0 192M Glycine 0 1 SDS pH 8 3 1X Three Buffer System for Semi Dry Electroblotting This buffer is used with the Galileo Semi dry Electroblotters Final 1X Composition Anode 1 Buffer 0 3M Tris Base 20 MeOH pH 10 4 Anode 2 Buffer 0 025M Tris base 20 MeOH pH 10 4 Cathode Buffer 0 025M Tris Base 0 04M Caproic Acid 20 MeOH pH 9 4 50X NAQ Northern Transfer Buffer 8 For transfer of RNA from agarose gels With its high buffering capacity and low ionic strength this buffer is more efficient than TAE TBE or MOPS for agarose gels 0 2M morpholinopropanesulfonic acid MOPS 50mM sodium acetate 5mM EDTA pH 7 0 50X NAQ Southern Transfer Transfer of DNA from agarose gels 1M ethanolamine glycine buffer pH 11 10X NAQ Transfer Buffer 0 8M Tris 1 18M borate 24mM EDTA pH 8 3 CAPS Buffer PH 11 This buffer can be used to improve transfer of some proteins 10mm CAPS 3 cyclohexylamino 1 10 methanol propanesulfuric acid adjust to pH 11 with sodium hydroxide DNA RNA If these gels were not run in 1XTBE they should be equilibrated for 10 minutes in this buffer Protein Gels After electrophoresis wearing gloves remove the gel assembly from the apparatus and place it under cold running water until the surfaces of both the
4. a squirt bottle may be used to aid separation of the gel from the glass plate Assembly of Transfer Stack Place the gel in blotting buffer and agitate gently Mark the membrane to indicate the side to which the samples will be on This is important in the event that any successive probe is negative and to indicate sample orientation Wet the membrane according to its manufacturers recommendations followed by a quick equilibration in transfer buffer Cut six 6 pieces of Extra Thick blotting paper and a piece of blotting membrane to the exact size of the gel to be blotted It is often helpful to have all the filter paper and membrane sitting in transfer buffer as you start to build the blotting sandwich Lay three 3 pieces of thoroughly soaked filter paper on the bottom anode electrode plate of the blotting unit Make sure that the pieces are lined up squarely Using a roller and only gentle pressure remove any air bubbles from between the papers Add a few ml of buffer to the papers and place the thoroughly soaked blotting membrane on top of the filters papers Add a few ml of buffer to the membrane remove the gel from the blotting buffer and place on top of the blotting membrane Be careful not to introduce any air bubbles Finally place three 3 thoroughly soaked filter papers on top of the gel Use a roller to gently remove any air bubbles from the stack Holding the stack drain off all excess buffer from the plate Wipe a
5. units in metal trays A Acrylamide is a volatile cumulative neurotoxin and suspected carcinogen Wear effective protective clothing and follow recommended handling and disposal procedures Polymerized gels contain some unpolymerized monomer Handle with gloves only A DO NOT move the unit when it is running B General Care amp Cleaning WARNING Acrylic is not resistant to aromatic or halogenated hydrocarbons ketones or esters Organic solvents cause acrylic to craze or crack Do not use ethanol or other organic solvents to clean your unit Do not autoclave bake or microwave your unit Before using clean and dry unit with DISTILLED WATER ONLY dry parts with clean tissues or air dry Do not use abrasive creams or cleaning brushes on the electrodes A thorough rinse with Distilled Water is all that is generally required to clean the unit after use A mild detergent may also be used Acrylic can also be exposed to a mild bleach solution 10 1 gt gt D Different Kinds of Blotting Western Blotting a blotting method for proteins that uses specific antibodies attached to a particular protein to help identify it It is often performed after SDS PAGE or some other form of polyacrylamide gel electrophoresis Southern Blotting a method sometimes called hybridization because a radioactive probe is hybridized or attached to specific pieces of DNA Northern Blotting similar to Southern but the molecules involved are
6. Galileo Bioscience User s Manual Semi Dry Electroblotters 91 1010 SD amp 91 2020 SD WARNING THESE UNITS ARE CAPABLE OF DELIVERING POTENTIALLY LETHAL VOLTAGE WHEN CONNECTED TO A POWER SUPPLY AND ARE TO BE OPERATED ONLY BY QUALIFIED TECHNICALLY TRAINED PERSONNEL PLEASE READ THE ENTIRE OPERATOR S MANUAL THOROUGHLY BEFORE OPERATING THIS UNIT Please check that the unit has been received complete and undamaged following shipment Any Faults or losses must be notified to Galileo immediately Galileo cannot accept responsibility for Goods returned without prior notification REFER TO PACKING LIST AND CHECK THAT ALL COMPONENTS AND ACCESSORIES WERE RECEIVED SAFETY PRECAUTIONS Please read the User Manual carefully before using the Semi Dry Electro Blotting Unit This manual contains important operating and safety information Our electrophoresis units are designed to perform flawlessly for years in the most demanding laboratories Please take the time to read the manual to ensure that you understand the safety and operating instructions to ensure the successful use of the unit Alterations could cause serious injury to the user or the system Only use the Galileo Semi Dry Blotter with a ground isolated external DC power supply The power supply should be equipped with No Load or Open Circuit protection Operating limits for the Galileo Semi Dry Blotter are Maximum Voltage Limit 50 Volts DC Operating Temperature 4 40 C A The Galileo Sem
7. Gemini Scientific reserves the right to alter the specifications of all Electrophoresis Systems without prior notice This will enable us to implement developments as soon as they arise If there has been shipping damage or incorrect items received please contact Gemini Scientific immediately Please do not return any goods without receiving a Return Authorization Introduction The Galileo Insight Semi Dry Electro Blotter enables rapid and highly efficient transfer nucleic acid or protein fragments from polyacrylamide or agarose to blotting membranes such as nitrocellulose nylon or PVDF membranes Electroblotting of gels is more efficient than capillary methods often taking less than an hour and is suitable for Southern northern or western blotting Insight semi dry electroblotter s solid plate platinized titanium and stainless steel electrodes are highly conductive and allow transfer at low voltages without external cooling systems The systems use large flat electrodes and consume significantly less transfer buffer without a loss in transfer efficiency or signal strength Once proteins nucleic acids in a sample aliquot have been separated on a slab gel the resulting bands may be transferred to a solid support membrane The primary reason for this type of blot is one of localization and secondarily concentration of discrete protein bands Although many have used alternative cross linking agents such as DATD N N dihydro
8. RNA Both Southern and northern blotting generally requires the DNA or RNA to first be separated out on an agarose gel Setting Up Power Supply Blotting requires a power supply than can operate at 10 V If an inappropriate power supply is used the power supply may blow a fuse shut itself off display a no load or short load message or even have a short circuit It is very important to be sure that the power supply you will be using will work with this device Blotting Buffer The most commonly used buffer for protein blotting from polyacrylamide gels is Towbin buffer Small amount of buffer may be needed for equilibrating the gel and membrane prior to blotting The buffer should be cooled to 4 C Filter Paper Whatman 3MM or equivalent is recommended Large sheets or rolls of can be cut to size and used Blotting Membrane Nitrocellulose for western or Southern blotting e PVDF polyvinylidene difluoride for western blotting Nylon for Southern or northern blotting See the following table for more details on each membrane Membrane Properties Applications Pore size Reprobing Nitrocellulose Most widely used membrane for Western transfer 0 2 um No western blotting Amino acid 0 45 um Good binding capacity analysis Proteins bind to the membrane due to Solid phase assay hydrophobic interactions systems Protein binding capacity 80 g cm PVDF Higher binding capacity than Protein 0 2 um Yes nitrocellulose Sequencing 0 45 u
9. ansfer performed for too short a time Solution Increase the amount of time for transfer Cause Transfer sandwich was assembled in the wrong order Solution The Insight Semi Dry Electroblotter is configured with the cathode on the top and the anode on the bottom This means that a downward transfer is being performed Follow instructions carefully when assembling the transfer sandwich Cause The pH of the transfer buffer is too close to the isoelectric point of the protein Solution Try a more acidic or basic transfer buffer Cause Too much methanol in the transfer buffer Solution Reducing methanol can help elute proteins from the gel but can reduce binding to nitrocellulose membranes Cause High percentage gels restrict transfer Solution Higher percentage acrylamide or crosslinker can restrict elution of proteins Use the lowest percentage acrylamide possible to separate your proteins Cause Puddles of buffer were present on the anode allowing the current to bypass the stack Solution Always clean the lower plate before placing the lid on the unit Do not squeeze the stack excessively as this also creates puddles that the current can pass through Cause The filter paper was too dry Solution Filter paper should be saturated with transfer buffer before adding them to the sandwich NITROCELLULOSE MEMBRANES PROBLEM Insufficient binding of proteins to t
10. glass plates are cool This facilitates handling of the gel and prevents the gel from curling Place the gel assembly with the notched or shorter plate facing up flat on paper towels on the lab bench and remove any excess liquid Remove one side spacer and insert a long metal spatula between the glass plates where the spacer had been Use care not to insert the blade close to the notches Gently rock the spatula forcing separation of the plate from the gel The gel will normally remain affixed to the bottom non notched plate Remove the top notched plate by slowly lifting it from the side with the inserted spatula and gradually increasing the angle until the plate is completely separated from the gel If the gel sticks to the top plate in an isolated spot a stream of water from a squirt bottle can be sprayed at the spot of separation Once the plates are separated remove the second side spacer along with any extraneous bits of acrylamide around the gel If excess water was utilized to aid in the separation of the gel from the glass use a paper towel to absorb the excess liquid Using a dry piece of dry blotting paper gently roll it onto the gel beginning at one end and working slowly towards the other end Care should be taken to prevent air bubbles from forming between the paper and the gel Beginning at one end of the gel slowly lift and peel back the paper from the glass with the gel attached As before a stream of water from
11. he membrane Cause Over transfer through the membrane Solution Use 0 2 micron pore size nitrocellulose instead of 0 45 micron or use PVDF with a higher binding capacity 14 Cause Not enough methanol in the transfer buffer Solution Nitrocellulose binds proteins best when 20 methanol is used in the transfer buffer Cause Low MW proteins are not binding well or are being washed away Solution Use glutaraldehyde to crosslink the proteins to the membrane and use Tween 20 in the wash steps Cause SDS is preventing binding Solution Eliminate SDS in the transfer buffer PVDF PROBLEM Smeared or swirled transfe r and missing bands Cause Membrane was dried out before it was added to the transfer sandwich Solution Membrane should be completely gray and slightly translucent when added to the sandwich If it has dried out re wet in methanol and equilibrate in transfer buffer Cause Alcohol was not used to pre wet the membrane Solution PVDF is hydrophobic and requires a short soak in methanol prior to transfer Cause Air spaces are interfering with contact between the gel and the membrane Solution Roll a test tube or pipet over the membrane make sure it is clean before putting the rest of the sandwich together Cause Electrophoretic conditions were incorrect or not ideal Solution Running conditions sample preparation pe
12. i Dry Blotter is designed to be operated with the lid in place If the lid is removed the electrical connection will be broken Do not attempt to use the blotter without the lid in place A Always disconnect the unit from the power supply to avoid the risk of personal shock Isolate the power supply from the main power source FIRST then disconnected the power cords A Running Conditions should not exceed the maximum operating voltage or current A Always disconnect the unit from the power supply when you want to move the unit or add running buffer A Use this apparatus only for its intended purpose as described in this manual Do not use this product if the power cords are damaged or if any of its surfaces are cracked Gemini Scientific Inc DBA Galileo Bioscience is not responsible for any injury or damage caused by operating this blotter in a manner not specified in this manual WARRANTY Gemini Scientific Inc DBA Galileo Bioscience guarantees that the Semi Dry Electro blotting System you have received has been thoroughly tested and meets its published specification This warranty is valid for 36 months only if the product and functions have been used according to the user manual No liability is accepted for loss or damage arising from incorrect use Gemini s liability is limited to the repair or replacement of the unit or refund of the purchase price at their option Gemini Scientific is not liable for any consequential damages
13. m Strong hydrophobic character and Western transfer solvent resistant Amino acid Physically stronger than Analysis nitrocellulose Solid phase assay Compatible with commonly used protein systems stains and immunodetection methods Protein binding capacity 50 150 g cm Nylon Microporous membrane modified Southern and 0 45 um Yes with strongly basic charged groups northern transfers Ideal for binding negatively charged Solid phase biomolecules such as DNA and RNA immobilization Low background for enhanced Dry chemistry resolution test strips Membrane is formed around a non woven Enzyme polyester fiber matrix which confers immobilization high tensile strength toughness Gene probe assays and flexibility RECIPES FOR BUFFERS 1X Tris Borate EDTA Buffer TBE 1X or 0 5X TBE is used for agarose gel electrophoresis and semi dry electroblotting of nucleic acids Final 1X Composition 89mM Tris Base 89mM Boric Acid 2mM Disodium EDTA pH 8 3 1X Towbin Buffer 1X Tris glycine buffer Towbin buffer minus the methanol is used for agarose and polyacrylamide gel electrophoresis of nucleic acids Towbin buffer containing 20 methanol is a commonly used buffer for semi dry transfers 0 025M Tris Base 0 192M Glycine 20 MeOH pH 8 3 1X Tris Acetate EDTA Buffer TAE 1X TAE is used for agarose and polyacrylamide gel electrophoresis and semi dry electroblotting of nucleic acids Final 1X Composition 0 04M Tris
14. rcentage acrylamide and many other variables can affect the migration and resolution of proteins Please review your electrophoretic conditions Cause Transferring at too higha current Solution Please refer to the Running Conditions in the manual Cause Membrane was not thoroughly wetted Solution Always pre wet the membrane according to the manufacturer s instructions White spots indicate dry areas of the membrane Cause Too much Current Solution Running at constant voltage can cause power fluctuations that will cause overheating A buffer that has not been made correctly or that has too high an ionic strength can also burn a gel by overheating A cracked and dry gel often is an indicator of overheating 15 The following is a chemical compatibility chart for the care of an acrylic device Although acrylic is compatible with most solvents and solutions found in a biochemical laboratory some solvents can cause damage This list does not include all possible chemical incompatibilities and safe compounds Acrylic products should be cleaned with warm water a mild detergent and can be exposed to a mild bleach solution 10 1 RNAse removal products are also safe for acrylic Chemical Acetic Acid 5 Acetic Acid Glacial Acetic Anhydride Acetone Ammonia Ammonium Chloride saturated Ammonium Hydroxide 10 Ammonium Hydroxide concentrate Aniline Benzene Butyl Ace
15. s Clinical Biotechnology 1 43 49 1989 This review article on Western Blotting gives a good overview of factors such as transfer buffers types of membranes and post membrane stains This was written for standard tank blotting but much of it is applicable to semi dry blotting Eckerskorn Chrisoph and Lottspeich Friedrich Structural Characterization of blotting membranes and the influence of membrane parameters for electroblotting and subsequent amino acid sequence analysis of proteins Electrophoresis 14 831 838 1993 A useful reference if you plan to do protein sequencing of transferred samples Legendre Nancy Immobilon P Transfer membrane Applications and Utility in Protein Biochemical Analysis BioTechniques supplement to Vol 9 788 805 1990 This reference deals specifically with transfer conditions using Immobilon P type membranes Tovey E R and B A Baldo 1987 Comparison of semi dry and conventional tank buffer electro transfer of proteins from polyacrylamide gels to nitrocellulose membranes Electrophoresis 8 384 387 This paper discusses quantitative yields of proteins of different molecular weights using different transfer conditions Dunbar B S Ed 1994 Protein Blotting A Practical Approach IRL Press at Oxford University Press Oxford England A great guide to blotting techniques including visualization immunological techniques and sequence analysis Nucleic Acid Blotting References 8 10
16. sorption of the liquid Slight crazing but acrylic has retained most of its strength Any Damage is mostly visual not structural UNSATISFACTORY Softened swollen slowly dissolved DISSOLVED In seven days of less Code 16 CHUN CF HN Oo nNnNnUDUVDVeETNNNHNHNHNHUNHNHANHNH NM
17. tate Calcium Chloride saturated Carbon Tetrachloride Chloroform Chromic Acid 40 Citric Acid 10 Cottonseed Oil edible Detergent Solution Heavy Duty Diesel Oil Diethyl Ether Dimethyl Formamide Dioctyl Phthalate Ethyl Acetate CODES Code NnnorFr DN OU FCC HNHNNNCT CNT VON Chemical Ethyl Alcohol 50 Ethyl Alcohol 95 Ethylene Dichloride Ethylene Glycol 2 Ethylhexyl Sebacate Formaldehyde 40 Gasoline regular leaded Glycerine Heptane commercial grade Hexane Hydrochloric Acid 10 Hydrochloric Acid concentrate Hydrofluoric Acod 40 Hydrogen peroxide 28 solution Hydrogen peroxide 3 solution Hydroxide 10 Isooctane Isopropyl Alcohol 100 Kerosene no 2 fuel oil Lacquer Thinner Methyl Alcohol 100 Methyl Alcohol 50 Methyl Ethyl Ketone Methylene Chloride Mineral Oil white SAFE No effect except some possible staining Code Nnnnooc gt nNnuoucrFCTNM SF NHN CTCUHNH NN Chemical Naptha Nitric Acid 10 Nitric Acid 40 Nitric Acid concentrate Oleic Acid Olive Oil Phenol 5 solution Soap Solution Ivory Sodium Carbonate 2 Sodium Carbonate 20 Sodium Chloride 10 Sodium Hydrochlorite 5 Sodium Hydroxide 1 Sodium Hydroxide 10 Sodium Hydroxide 60 Sulfuric Acid 3 Sulfuric Acid 30 Sulfuric Acid concentrate Toluene Trichloroethylene Turpentine Water distilled Xylene ATTACKED Slight attack by or ab
18. way any droplets around the edges of the stack Running the Blot Place the lid on the unit and loosely tighten down the lid with the supplied black knobs The weight of the lid is usually enough and the tightening is not required Note Do not tighten for gels thicker than 1 5mm or when using more than six 6 filter pads total If the screws are used tip the unit on an angle to drain off any excess buffer that may have been squeezed out The buffer will remain safely in the moat around the electrode plate Attach the power leads red to red and black to black to an appropriate power supply The black lead has a acrylic shroud that will stop it from attaching to the red anode Transfer Settings Blotting takes place at a given migration rate for a specified time The units are mA times hrs If you need to slow the transfer down to say coincide with the setting up of a probe simply decrease the current mA to match the added time you require mA hr Std Setting mA hr New Setting alternatively you can increase the current to decrease the time This assumes that you have determined an initial mAh value that works well for the molecules you are interested in A current to use for a 45 minute time period is based on the area cm cm of your gel The range is 0 8 to 2 mA per square centimeter of gel For example the area of a 10 x 10 cm gel would be 100 cm the current range would be 80 mA 0 8mAcm x 100cm to 200 mA 2mAcm
19. x 100cm Blots may also be run at a constant voltage Some power supplies have difficulty sustaining steady voltages at these low voltage settings If you find that voltages are fluctuating or that the power supply shuts itself off when set on constant voltage use constant current settings instead Read your power supply s instructions to ensure that the power supply will work at a voltage lower than 20V These voltages often occur in semi dry electroblotting Contact the manufacturer regarding the unit s performance under high current low voltage conditions if you have any questions 10 Factors That Affect Transfer Efficiency While general conditions can be described which will result in successful transfer of most molecules it should be noted that optimal transfer conditions will vary based on the characteristics of the molecule you are working with Some factors that affect transfer rate and efficiency include molecule size charge gel thickness and percentage and hydrophobicity The reference list at the end of the manual provides useful information that can help you choose optimal conditions for efficient transfer of a specific molecule RUNNING CONDITIONS Protein DNA RNA Membrane Nitrocellulose 45 or 0 2u Nylon PVDF 0 45u or 0 2u Transfer Buffer Towbin Buffer 3 Buffer 0 5X 1XTBE TAE NAQ System Bjerrum and Schafer Nielsen Power Settings Constant current 0 8 3mA per Constant Current 0 5
20. xyethylene bis Acrylamide to allow for the accessibility of gel bound proteins this still represents an impediment to radio enumeration due to the quenching by the gel matrix itself The most common solid support membrane is nitrocellulose A second type of membrane is PVDF Polyvinylidene difloride which is generally used when a transferred protein is to be sequenced additionally it has a 2x binding capacity Also used for nucleic acid capture are Nylon membranes In either case the proteins are transferred from the gel to the matrix in an electric field perpendicular to the gel initial running direction Tris based buffers are employed in the transfer Methanol and SDS are modifiers often used in protein transfer buffer These components however are antithetical in their effects both in terms of movement and absorption Methanol restricts protein movement from the gel but is often required to support the ionic nature of protein to nitrocellulose binding SDS aids in protein elution but can also inhibit binding of small molecular weight proteins Mozdzanowski J High yield electroblotting Electrophoresis 1992 Vol 13 p 59 64 Using Semi Dry Electro Blotter Units A SAFETY PRECAUTIONS A READ all instructions before using the unit A Always turn off power supply FIRST then disconnect the power cords Always have electrophoresis unit disconnected from their power supply before removing the safety cover A DO NOT operate electrophoresis
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