Please read manual carefully before starting experiment
Contents
1. model cell line protein tyrosine kinase inhibitors growth factors or cytokines Microplate reader capable of measuring absorbance at 450 nm 37 incubator Precision pipettes to deliver 2 ul to 1 ml volumes Adjustable 1 25 ml pipettes for reagent preparation 100 ml and 1 liter graduated cylinders Absorbent paper Distilled or deionized water Orbital shaker or oscillating rocker OS A A U 4 RayBio Cell Based GSK 3a S21 ELISA Kit Protocol IV REAGENT PREPARATION NOTE Thaw all reagents to room temperature immediately before use If wash buffers contain visible crystals warm to room temperature and mix gently until dissolved NOTE Briefly centrifuge 1 000g ITEMS G and before opening to ensure maximum recovery ITEM COMPONENT PREPARATION EXAMPLE A Uncoated 96 Well Microplate No Preparation N A 20X Wash Buffer A Concentrate pilute each 20 fold with distilled 1 of concentrate 475 ml of detonized wat water 500 ml of 1X working 20X Wash Buffer B Concentrate A ea sen solution Fixing Solution No Preparation N A 1 ml of trate 29 ml of 30X Quenching Buffer Dilute 30 fold with 1X Wash Buffer MES E wash buffer 30 ml of 1X Concentrate A s working solution 20 ml of concentrate 80 ml of F 5X Blocking Buffer Concentrate Dite Ss e HO IS UC OF water 100 ml of 1X working deionized water j solution 14ul of trate 6986 ul of2. To avoid cell loss do not pipette directly onto the cells Instead gently dispense the liquid down the wall of cell culture wells Also avoid the use of vacuum suction or too forcefully tapping the microplate when discarding any solution 6 Add 100 ul of Fixing Solution ITEM D into each well and incubate for 20 minutes at room temperature NOTE The fixing solution is used to permeabilize the cells 7 Repeat wash step 5 7 RayBio Cell Based GSK 3a S21 ELISA Kit Protocol 8 Add 200 ul of the prepared 1X Quenching Buffer ITEM E into each well and incubate 20 minutes at room temperature NOTE The quenching buffer is used to minimize the background response 9 Wash 4 times with 1X Wash Buffer A 10 Add 200 ul of the prepared 1X Blocking Buffer ITEM F into each well and incubate for 1 hour at 37 C 11 Wash times with the prepared 1X Wash Buffer B ITEM C NOTE If needed the microplate may be stored at 80 C for several days after this wash 12 Add 50 ul of the prepared 1X primary antibody ITEM G or H into each corresponding well and incubate for 2 hours at room temperature 13 Wash 4 times with 1X Wash Buffer 14 Add 50 ul of 1X HRP Conjugated Anti Mouse IgG ITEM 1 against Mouse Anti GSK 3a ITEM H and 1x HRP Conjugated Anti Rabbit IgG ITEM J against Rabbit Anti phospho S21 GSK 3a ITEM G into corresponding wells and incubate for 1 hour at room temperature 15 Wash 4 times with 1X Wash Bu
3. 1 ELISA kit is a very rapid convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in cells It can be used for measuring the relative amount of GSK 3a S21 phosphorylation and screening the effects of various treatments inhibitors such as siRNA or chemicals or activators in cultured human and mouse cell lines By determining GSK 3a protein phosphorylation in your experimental model system you can verify pathway activation in your cell lines without spending excess time and effort in preparing cell lysate and performing an analysis of Western Blot In the Cell Based GSK 3a S21 ELISA kit cells are seeded into a 96 well tissue culture plate The cells are fixed after various treatments inhibitors or activators After blocking Anti Phospho GSK 3a S21 or Anti GSK 3a is pipetted into the wells and incubated The wells are washed and HRP conjugated anti mouse or anti rabbit IgG is added to the corresponding wells The wells are washed again a TMB substrate solution is added to the wells and color develops in proportion to the amount of protein The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm See Figure 1 below for an illustration 2 RayBio Cell Based GSK 3a S21 ELISA Kit Protocol Il HOW IT WORKS 1 Add cells 2 Treatment with stimulators 3 Fixing and blocking or inhibitors Lee Li ll 4 Anti
4. 500X Rabbit Anti phospho S21 Dilute 500 fold with 1X Blocking WW Eee gt G 1X Blocking Buffer 7ml of 1X za GSK 3a Concentrate Buffer E lt 9 working solution ty 7 of concentrate 6993ul of 1X cz j x Blocking Buffer 7ml of 1x Concentrate gt working solution 8 Anti Mouse IgG Concentrate Buffer 10 ul of concentrate 9990 pl of z 1X Blocking Buffer 10 ml of 1X 1000X HRP Conjugated working solution lt Anti Rabbit IgG Concentrate K TMB Substrate No Preparation N A L Stop Solution 5 RayBio Cell Based GSK 3a S21 ELISA Kit Protocol V ASSAY PROCEDURE NOTE ALL incubations and wash steps must be performed under gentle rocking or rotation 1 2 cycles sec 1 Design your experiment For example see Figure 2 below PDGF bb ng ml 0 20 100 0 20 100 0 20100 0 20 100 O O O O O 0 min 88 OUO 10 min OOOO OO Q2 O OOO 20 min vn CC OTIO OO HO CHO CE C C VOC RP NO OOK E E E Anti Phospho Anti GSK 3a Inhibitor Anti Inhibitor GSK 3a S21 Phospho GSK 3a Anti GSK 3a S21 Fig 2 Example of how to seed cells for RayBio cell based assay OPTIONAL If seeding HUVECs HMEC 1 or other loosely attached cells coat the Uncoated 96 Well Microplate ITEM A by adding 100 ul poly L Lysine Recommended Sigma Aldrich P4832 into each well and then follow m
5. EW ej Cell Based Human Mouse GSK 3a S21 Phosphorylation ELISA Kit For the semi quantitative detection of phosphorylated human or mouse GSK 3a S21 and total GSK 3a in adherent whole cell lines User Manual Revised March 6 2015 Cat CBEL GSK3A 1 1 plate kit Cat CBEL GSK3A 2 2 plate kit Cat CBEL GSK3A 5 5 plate kit Please read manual carefully before starting experiment Ke RayBiotech The protein array pioneer Tel Toll Free 1 888 494 8555 or 1 770 729 2992 Fax 1 770 206 2393 Website www raybiotech com Email info raybiotech com Cell Based Human Mouse GSK 3a S21 Phosphorylation ELISA Kit TABLE OF CONTENTS Passe 2 Il How It DXIMDMIDMDMDMDMDMDMDMBMMIMMBN 3 IIl Reagents and Storage 4 IV Additional Reagents Required 4 V Reagent Preparation 5 Assay Procedure mnnn 6 VII Assay Procedure Summary 9 VIII Quality Control Data 10 IX Troubleshooting Guide 12 1 RayBio Cell Based GSK 3a S21 ELISA Kit Protocol INTRODUCTION Protein phosphorylation is instrumental in the regulation of protein activity within a cell It plays important roles in the living cells including proliferation differentiation and metabolism A large number of protein kinases and phosphatases have been extensively investigated and have been shown to be involved in signal transduction pathways The RayBio Cell Based Human Mouse GSK 3a S2
6. anufacturer s instructions A pre coated CellBIND microplate or other poly lysine treated tissue culture plate may be used in place of Item A 6 RayBio Cell Based GSK 3a S21 ELISA Kit Protocol 2 Seed 100 ul of 30 000 cells into each well of the Uncoated 96 Well Microplate ITEM A provided and incubate overnight at 37 C with 5 NOTE The optimal cell number used will vary on the cell line and the relative amount of protein phosphorylation More or less cells may be used but this must be determined empirically NOTE The cells can be starved 4 24 hours depending on cell line prior to treatment with inhibitors or activators 3 Apply various treatments inhibitors such as siRNA or chemicals or activators according to manufacturer s instructions and incubate for the desired time points NOTE It is recommended to dissolve inhibitors or activators into serum free cell culture medium before treating the cells unless otherwise stated in the manufacturer s instructions 4 Discard the cell culture medium by flipping the microplate upside down and gently tapping the bottom of the microplate over a sink 5 Wash by pipetting 200 ul of the prepared 1X Wash Buffer A ITEM B into each well Discard the wash buffer same as step 4 and wash 2 more times for a total of 3 washes using fresh wash buffer each time After the final wash gently blot the microplate onto a paper towel to remove any excess remaining buffer NOTE
7. ffer B 16 Add 100 ul of the TMB Substrate ITEM into each well and incubate for 30 minutes at room temperature in the dark 17 Add 50 ul of the Stop Solution ITEM L into each well Read at 450 nm immediately 8 RayBio Cell Based GSK 3a S21 ELISA Kit Protocol ASSAY PROCEDURE SUMMARY 1 Seed 30 000 cells into each well and incubate overnight 2 Apply various treatment inhibitors or activators according to manufacturer s instructions 3 Add 100 ul of Fixing Solution into each well and incubate for 20 minutes at room temperature 4 Add 200 ul of prepared 1X Quenching and incubate for 20 minutes at room temperature J 5 Add 200 ul of prepared 1X Blocking Buffer and incubate for 1 hour at 37 C 6 Add 50 ul of prepared 1X primary antibody to each well and incubate for 2 hours at room temperature J 7 Add 50 ul of prepared 1X HRP Conjugated secondary antibody and incubate for 1 hour at room temperature J 8 Add 100 ul TMB Substrate and incubate 30 minutes at room temperature 9 Add 50 ul Stop Solution to each well Read at 450 nm immediately 9 RayBio Cell Based GSK 3a S21 ELISA Kit Protocol VII QUALITY CONTROL DATA Representative results of Cell Based GSK 3a S21 are shown below 1 Seeded 30 000 cells into appropriate wells of the microplate Cells were incubated at 37 C in 5 overnight 2 Added 50 ul of different conce
8. mmendation for washing Reduce the cell number 1 Inaccurate pipetting 3 Large CV 2 Remaining wash buffer in the well 3 Cells drop off from the wells Check pipette Remove all of wash buffer Please don t directly face the cells with tips when adding reagents or wash buffer 12 RayBio Cell Based GSK 3a S21 ELISA Kit Protocol Note 13 RayBio Cell Based GSK 3a S21 ELISA Kit Protocol Note 14 RayBio Cell Based GSK 3a S21 ELISA Kit Protocol This product is for research use only 2015 RayBiotech Inc 15 RayBio Cell Based GSK 3a S21 ELISA Kit Protocol
9. ntrations of stimulators rhPDGF bb concentration for NIH3T3 cells 0 or 50 ng ml in serum free DMEM to appropriate wells shown below Then incubated for 10 min at 37 C 3 Discarded the solution and wash 3 times with 1X Wash Buffer A 200 ul each immediately Then flipped the plate upside down and tapped to remove all of excess wash buffer The protocol was then followed as stated 10 RayBio Cell Based GSK 3a S21 ELISA Kit Protocol Unireat 7 Treat OD 450nm Phospho Pan GSK3a Fig 3 NIH3T3 cells were stimulated by 50ng ml of recombinant human PDGFbb for 10 minutes at 37 PDGFBB 0 10 0 10 Min Anti GSK 3a 821 Anti pan GSK 3a Fig 4 NIH 3T3 cells were treated or untreated with 50 ng ml recombinant human PDGFbb for 10 min Cell lysates were analyzed using Western Blot 11 RayBio Cell Based GSK 3a S21 ELISA Kit Protocol IX TROUBLESHOOTING GUIDE Problem Cause 1 Improper storage of 1 Low signal the ELISA kit 2 Improper dilution 3 Cells drop off from the wells Solution 1 Store the kit according to manual instructions Keep substrate solution in dark Ensure correct preparation of antibody and reagents Some of treatments may make cells drop off the wells Reduce inhibitor or activator concentration 1 Inadequate washing 2 High background 2 Too much cells a Be sure to remove all of washing solution and follow the reco
10. phospho protein antibody 5 HRP conjugated secondary 6 Develop with substrate or anti pan protein antibody antibody TMB Color R Je k 7 WW LAC Fig 1 Cell Based protein phosphorylation procedure 3 RayBio Cell Based GSK 3a S21 ELISA Kit Protocol Ill REAGENTS AND STORAGE Store entire kit at lt 20 C immediately upon arrival Kit must be used within the 6 month expiration date Avoid repeated freeze thaw cycles STORAGE AFTER ITEM COMPONENT 1 PLATE KIT 2 PLATE KIT INITIAL THAW A Uncoated 96 Well Microplate 1 plate 2 plates Room Temperature 20X Wash Buffer A Concentrate 1 vial 30 ml C 20X Wash Buffer B Concentrate 1 vial 30 ml 2 8 C D Fixing Solution 1 vial 30 ml E 30X Quenching Buffer Concentrate 1 vial 2 ml F 5X Blocking Buffer Concentrate 1 vial 20 ml 2 8 C 1 month 500X Rabbit Anti phospho S21 GSK i 1 vial 14 pl 2 vials 14 ul ea 1000X M Anti GSK 3 H STD 1 vial 73 2 vials 7ul ea Concentrate 20 C 1000X HRP Conjugated Anti Mouse IgG Concentrate a Mato EL 2 1000 HRP Conjugated i Anti Rabbit IgG Concentrate 2 vials tO alee TMB Substrate 1 vial 12 ml 2 vials 12 ml ea e L Stop Solution 1 vial 14 ml For up to 3 months unless otherwise stated or until expiration date Contains 0 2 M Sulfuric Acid Ill ADDITIONAL MATERIALS REQUIRED 1
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