Manual - RayBiotech, Inc.
Contents
1. solution during experiment Sample is too concentrated Increase sample dilution RayBio Human Heat Shock 13 Protein Antibody Array Kit RayBiotech Inc the protein array pioneer company strives to research and develop new products to meet demands of the biomedical community RayBio patent pending technology allows detection of over 1000 cytokines chemokines and other proteins in a single experiment Our format is simple sensitive reliable and cost effective Products include Cytokine Arrays Chemokine Arrays ELISA kits EIA kits Phosphotyrosine kits Recombinant Proteins Antibodies and custom services This product is for research use only 2009 RayBiotech Inc RayBio Human Heat Shock 14 Protein Antibody Array Kit2. prepare a 100X Protease Inhibitor Cocktail Concentrate Note Be sure to complete 2X Cell Lysis Buffer preparation before proceeding to 3 3 20X Wash Buffer or Il If the Wash Buffer Concentrate 20X contains visible crystals warm to room temperature and mix gently until dissolved Dilute 20 ml of Wash Buffer Concentrate into 380 ml of deionized or distilled water to yield 400 ml of 1X Wash Buffer 4 Detection Antibody Cocktail Briefly spin the vial before use Add 150 ul of Blocking Buffer to the tube Mix gently and transfer the entire mixture to a tube containing 1 80 ml of Blocking Buffer to prepare 1X Cocktail of Biotin Conjugated Antibody Mix 5 1000X HRP Conjugated Streptavidin briefly spin down the HRP Streptavidin Concentrate to remove any liquid from the vial cap and pipette up and down to mix gently before use Prepare 1X HRP Conjugated Streptavidin Example add 5 ul of MHRP Conjugated Streptavidin concentrate into a tube with 4 995 ml Blocking Buffer and RayBio Human Heat Shock 6 Protein Antibody Array Kit mix gently Use immediately do not store the 1X Streptavidin for next day use Note mix tube containing 1 000X HRP Conjugated Streptavidin well before use since precipitation may form during storage V Overview and General Considerations A Preparation of Samples Remove supernatant from cell culture for attached cells wash cells twice with cold 1X PBS for suspension cells pellet the cells by spinnin
3. RayBio C Series Human Heat Shock Protein Antibody Array User Manual Cat AAH HSP 1 2 AAH HSP 1 4 AAH HSP 1 8 For Simultaneous Detection of the Relative Levels of 9 Heat shock protein Markers Version 1 0 August 31 2013 Please read manual carefully before starting experiment RayBiotech Inc We Provide You with Excellent Protein Array Systems and Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 1 888 547 0580 Website www raybiotech com Email info raybiotech com JUDIE HOr RayBiotech Inc RayBio Human Heat Shock Protein Antibody Array Kit TABLE OF CONTENTS Nis MIRO CIEE TION eosina 2 Il Principle of the ASSay ccccseecssseece eee 3 Ill Kit Components amp Storage cceee 5 IV Reagent Preparation ccsesssccceceeees V Overview and General Considerations a Preparation of Samples 00008 7 b Handling Array Membranes 06 8 Ce cubao henna 8 Vi Prologo lecura 8 a Blocking and Incubation 00 8 b Detection sessesessssesesreresesserssreseese 10 VII Interpretation of Results 11 VIL Array WIAD weiscscasscncatsncecndecsantaoveunaasconedexnoatonse 12 IX Troubleshooting Guide cceseeeeees 14 RayBio Human Heat Shock 1 Protein Antibody Array Kit l Introduction Heat shock proteins HSP are a family of functionally related molecular chaperones which play critical roles in protein foldi
4. fer D 1 vial 1 5 ml 1 vial 1 5 ml 1 vial 2 5 ml Protease Inhibitor 1 vial 1 vial 2 vials lt 20 C Cocktail 8 Well Incubation Tray 10 artid 1 tray 11 Plastic Sheets 4 sheets Room Temperature 12 Array Map Template 1 template 13 User Manual 1 booklet Each plastic package contains 2 or 4 membranes For up to 3 months unless stated otherwise or until expiration date Additional Materials Required Pipettors pipet tips and other common lab consumables Orbital shaker or oscillating rocker Tissue Paper Blotting Paper or Chromatography Paper Adhesive Tape or Plastic Wrap Distilled or De ionized Water Chemiluminescent blot documentation system CCD Camera X Ray Film and a suitable film processor Gel documentation system or other chemiluminescent detection system capable of imaging a Western blot RayBio Human Heat Shock Protein Antibody Array Kit IV Reagent Preparation 1 1X Cell Lysis Buffer 2x Cell lysis buffer should be diluted 2 fold with deionized or distilled water before use Note Proceed to 2 before completing the rest of the 2X Cell Lysis Buffer preperation Add 20 ul of prepared 100X Protease Inhibitor Cocktail Concentrate bring the tube to room temperature to thaw the solution before use into 1 98 ml 1X Lysis Buffer before use Mix well 2 Protease Inhibitor Cocktail Briefly spin down the Protease Inhibitor Cocktail tube before use Add 60 ul of 1X Lysis Buffer into the vial to
5. g down the cells at 1500 rpm for 10 min making sure to remove any remaining PBS before adding Lysis Buffer Solubilize the cells at 2x10 cells ml in 1X Lysis Buffer containing Protease Inhibitor Cocktail prepared in step 1 of Reagent Preparation Pipette up and down to resuspend cells and rock the lysates gently at 2 8 C for 30 minutes Transfer extracts to microfuge tubes and centrifuge at 14 000 x g for 10 min It is recommended that sample protein concentrations be determined using a total protein assay such as the BCA Assay Before applying to the membranes all lysates should be diluted at least 5 fold with 1X Blocking Buffer For the RayBio Human Heat Shock Protein Antibody Array Kit 200 ug ml to 600 ug ml of total protein from cell lysates after dilution with Blocking Buffer should be used for incubation Lysates should be used immediately or aliquotted and stored at 80 C Thawed lysates should be kept on ice prior to use Note If you experience high background you may further dilute your samples RayBio Human Heat Shock 7 Protein Antibody Array Kit B Handling Array Membranes e Always use forceps to handle membranes and grip the membranes by the edges only Flat tipped forceps are best for handling membranes e Never allow array membranes to dry during experiments e Avoid touching array membrane by hand tips or any sharp tools C Incubation e Completely cover membranes with sample or buffer d
6. in off excess detection reagent by holding the membrane vertically with forceps and touching the edge against a tissue Gently place the membrane protein side up on a piece of plastic sheet mark is on the protein side top left corner Cover the array with another piece of plastic sheet Gently smooth out any air bubbles Avoid using pressure on the membrane 3 Detect signal directly from membrane using chemiluminescence imaging system or expose to x ray film we recommend Kodak X Omat AR film to detect signal using film developer Expose the membranes for 40 Seconds Then re expose the film according to the intensity of signals If the signals are too strong background too high reduce exposure time e g 5 30 seconds If the signals are too weak increase exposure time eg 2 20 min or overnight Or re incubate RayBio Human Heat Shock 10 Protein Antibody Array Kit membranes overnight with 1X HRP conjugated Streptavidin and repeat detection on the second day 4 Save membranes at 20 C to 80 C for future reference VII Interpretation of Results Membrane signals are normally detected by a chemiluminescene imaging device Membranes also can be exposed to Kodak X Omat film at room temperature A biotinylated protein provides positive signals which can be used to identify the orientation and to normalize the results from different wells being compared By comparing the signal intensities relative expres
7. ng intracellular trafficking of proteins and coping with proteins denatured by heat shock and other stresses HSPs are found in virtually all living organisms from bacteria to humans HSP family includes 5 major classes according to their molecular weights i e the small heat shock proteins SHSPs HSP33 HSP60 HSP70 and HSP90 HSP100 The smaller 8 kD protein ubiquitin which marks proteins for degradation and is regarded as co chaperone also belongs to HSP family In addition to their roles in protein trafficking and and stress response HSPs are also important in cardiovascular functions and modulation of immune responses Some HSPs have been under investigation as therapeutic targets in cancer RayBio Human Heat Shock 2 Protein Antibody Array Kit Il Principle of the Assay With the RayBio Heat Shock Protein Antibody Array kit researchers can now simultaneously detect the relative level of 9 HSP related proteins in cell lysates By monitoring the changes in protein levels in different experimental model systems researchers can study pathway activation without spending excess time and effort in performing immunoprecipitations and or Western Blotting Each array membrane is pre printed with capture antibodies treated or untreated cell lysate is then added toeach membrane After extensive washing the membranes are incubated with a cocktail of biotin conjugated anti apoptotic protein antibodies After incubation with HRP Strep
8. om both untreated and induced HepG2 cells were incubated overnight with RayBio Human Heat Shock Protein Antibody Array membranes Control membrane was incubated with blocking buffer The membranes were then washed and cocktail of biotinylated antibodies was used to detect bound proteins After incubation with HRP streptavidin the signals were visualized by cherniluminescence RayBio Human Heat Shock 12 Protein Antibody Array Kit IX Troubleshooting Guide Problem Cause Recommendation Weak or no signal Taking too much time for detection Entire detection process must be completed in 30 minutes Film developer does not work properly Fix film developer Did not mix HRP streptavidin well before use Mix tube containing HRP streptavidin well before use since precipitates may form during storage Sample is too dilute Increase sample concentration Other Reduce blocking concentration by diluting in 1X Wash Buffer II Slightly increase HRP concentration Slightly increase biotin antibody concentration Expose film overnight Uneven signal Bubbles formed during incubation Remove bubbles during incubation Membranes were not completely covered by solution Completely cover membranes with solution High background Exposure to x ray film is too long Decrease exposure time Membranes were allowed to dry out during experiment Completely cover membranes with
9. sion levels of target proteins can be made The intensities of signals can be quantified by densitometry The positive controls can be used to normalize the results from different membranes being compared Antibody affinity to its target varies significantly between antibodies The intensity detected on the array with each antibody depends on this affinity therefore signal intensity comparison can be performed only within the same antibody antigen system and not between different antibodies One important parameter is the baseline signal response To obtain the best results we suggest that several exposures be attempted We also strongly recommend using a negative control in which the sample is replaced with an appropriate mock buffer according to the array protocol particularly during your first experiment RayBio Human Heat Shock 11 Protein Antibody Array Kit Vill Human Heat Shock Protein Antibody Array Map POSA NEG HSP27 HSP32 HSP40 HSP60 HSP70 HSP90 GRP75 NEG NEG POSA X Representative Data using Human Heat Shock Protein Antibody Array Buffer Only Untreated Cell Induced Cell e 2 08 8 V Teg i s e s gt e a gt e POSA HSP27 HSP32 HSP40 HSP60 HSP70 Rs ss e S S O HSP90 GRP75 UBIO HSP10 POSA Rs es es DD I Heat shock protein profiling in induced HepG2 cultured cells HepG2 cells were treated with 100 uM hydrogen peroxide for 24 hours 500 pg of cell lysates fr
10. tavidin the signals are visualized by chemiluminescence RayBio Human Heat Shock 3 Protein Antibody Array Kit How It Works YYYYY Incubation of Sample with i Y Y Y Y antibody array chips Overnight Antibody array chips K Cocktail of biotin conjugated anti ss HSP marker h xt 2 hrs Incubation with cocktail of biotin conjugated anti HSP marker Labeled Streptavidin Incubation with labeled 2hrs al Streptavidin ra h Data analysis RayBio Human Heat Shock 4 Protein Antibody Array Kit Ill Kit Components amp Storage Store entire kit at lt 20 C immediately upon arrival Kit must used within the 6 month expiration date STORAGE TEM COMPONENT 2 SAMPLE KIT 4 SAMPLE KIT 8 SAMPLE KIT TEMPERATURE AFTER THAWING 1 Human HSP Array C1 2 membranes 4membranes 8membranes lt 20 C 2 Blocking Buffer 1vial 25 ml 1vial 25 ml 2 vials 25 ml ea Detection Antibody f 2 8 C 3 Cocktail tya Na a up to 3 days after dilution 1 000X HRP Streptavidi i eee bie ere 1 vial 50 pl Concentrate 20X Wash Buffer 5 TN 1 vial 10 ml 1 vial 10 mi 1 vial 20 mi Concentrate Hall Rah DANSEN 1 vial 10 mi 1 vial 10 ml 1 vial 20 ml see Concentrate j 2X Cell Lysis Buffer 1 vial 6 mi Concentrate 8 Detection Buffer C 1 vial 1 5 ml 1 vial 1 5 ml 1 vial 2 5 ml 9 Detection Buf
11. ure with shaking 5 min per wash 5 Wash 2 times with 1 5 ml of 1X Wash Buffer II at room temperature with shaking 5 min per wash 6 Carefully remove wash buffer from each well containing array membranes 7 Add 1 ml of diluted Cocktail of Biotin Conjugated Antibody Mix to each membrane Incubate at 2 8 C with gentle shaking overnight Note Incubation may be done at room temperature for 2 hours 8 Wash as directed in steps 4 5 and 6 9 Add 1 5 ml of 1X HRP conjugated Streptavidin to each membrane 10 Incubate HRP streptavidin at room temperature for 1 5 hours Note incubation may be done overnight at 4 C RayBio Human Heat Shock 9 Protein Antibody Array Kit 11 Wash as directed in steps 5 and 6 B Detection NOTE Do not let the membrane dry out during detection The detection process must be completed within 40 minutes without stopping 1 Proceed with detection reaction Add 250 ul of Detection Buffer C and 250 ul of Detection Buffer D for one membrane mix both solutions Drain off excess wash buffer by holding the membrane vertically with forceps Place membrane protein side up mark is on the protein side top left corner on a clean plastic sheet provided in the kit Pipette the mixed Detection Buffer on to the membrane and incubate at room temperature with gentle shaking for 2 minutes Ensure that the detection mixture is completely and evenly covering the membrane without any air bubbles 2 Dra
12. uring incubation and cover eight well tray with lid to avoid drying e Avoid bubbles and foaming during incubation steps e Perform all incubation and wash steps under gentle shaking 1 2 cycles sec e The following incubations may be done overnight at 4 C all wash steps Step 3 sample incubation Step 7 biotin Ab incubation and Step 10 HRP streptavidin incubation VI Protocol A Blocking and Incubation 1 Place each membrane into the provided 8 well tray top left corner marked with Note The printed side should be facing upward 2 Add 1 ml Blocking Buffer and incubate at room temperature with gentle shaking for 30 minutes to block membranes 3 Decant Blocking Buffer from each well Add 1 ml diluted sample onto each array membrane and cover with the lid Incubate at 2 8 Ka RayBio Human Heat Shock 8 Protein Antibody Array Kit overnight on a rocker or shaker low speed 1 2 cycles sec Dilute sample using Blocking Buffer Note 1 Dilute cell lysates at least 5 fold with Blocking Buffer to avoid high background Note 2 Optimal sample dilution factors should be determined empirically More sample can be used if signals are too weak If signals are too strong the sample can be diluted further Note 3 Incubation may be done at room temperature for 4 hours but this may cause lower signals 4 Decant the samples from each well and wash 3 times with 1 5 ml of 1X Wash Buffer at room temperat
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