User Manual - RayBiotech, Inc.
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1. 3 0 25 mmm NIH3T3 me NIH3T3 PDGF E 2 04 c i Q 1 5 rt a O 1 0 0 5 4 M Phospho Akt Ser473 Pan Akt 10 RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol B Western Blot Analysis PDGF 0 10 0 10 Min Anti phospho Akt Anti pan Akt Ser473 iii SENSITIVITY The NIH3T3 cells were treated with recombinant human PDGF for 10 minutes to induce phosphorylation of Akt Serial dilutions of lysates were analyzed in this ELISA ELISA ODz450 nm 1 6 1 4 1 2 4 1 0 0 8 4 0 6 0 4 4 0 2 no B EH m 40 133 4 4 1 48 0 49 0 ug RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol X REFERENCES 1 Hajduch E et al 2001 FEBS Lett 492 199 203 2 Burgering B M and Coffer P J 1995 Nature 376 599 602 3 Franke T F et al 1997 Cell 88 435 7 12 RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol XI TROUBLESHOOTING GUIDE dilution Problem Cause Solution 1 Sample signals a Too low a Sample concentration is a Increasing sample too low concentration b Too high b Sample concentration is b Reducing sample too high concentration 2 Large CV a Inaccurate pipetting a Check pipettes 3 High background a Plate is insufficiently a Review the manual washed for proper washing If using an automated plate washer check that all ports are unobstructed b Contaminated2. 1 Prepare all reagents samples and standards as instructed 2 Add 100 ul sample or positive control to each well 8 RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol Incubate 2 5 hours at room temperature or over night at 4 C J 3 Add 100 ul prepared primary antibody to appropriate well Incubate 1 0 hours at room temperature 4 Add 100 ul prepared 1x HRP conjugated secondary antibody solution to corresponding well Incubate 1 hour at room temperature i 5 Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature J 6 Add 50 ul Stop Solution to each well Read at 450 nm immediately IX TYPICAL DATA ELISA data analysis Average the duplicate readings for each sample or positive i Positive Control A431 cells were treated with recombinant human EGF at 37 C for 20 min Solubilize cells at 4 x 10 cells ml in Cell Lysate Buffer Serial dilutions of lysates were analyzed in this ELISA Please see step 3 of Part VI Reagent Preparation for detail 9 RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol Assay Diluent OD 450 nm 0 1 T T T P 1 P 2 P 3 P 4 0 Positive control dilution series ii Recombinant Human PDGF Stimulation of NIH3T3 Cell Lines NIH3T3 cells were treated or untreated with recombinant human PDGF for 10 min Cell lysates were analyzed using this phosphoELISA and Western Blot
3. Assay Diluent 150u 150 150 TS Xy Xy Sy 35555 Pd P 2 P 3 P 4 0 4 If the Wash Concentrate 20x Item B contains visible crystals warm to room temperature and mix gently until dissolved Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer 5 Briefly spin the detection antibody Item C 1 or Item C 2 before use Add 100 ul of 1x Assay Diluent into the vial to prepare a detection antibody concentrate Pipette up and down to mix gently the concentrate can be stored at 4 C for 5 days or at 80 C for one month The anti phospho Akt Ser473 or anti pan Akt antibody should be diluted 55 fold with 1x Assay Diuent and used in step 4 of Part VII Assay Procedure 6 Briefly spin the HRP conjugated anti rabbit IgG Item D 1 6 RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol before use Pipette up and down to mix gently HRP conjugated anti rabbit IgG concentrate should be diluted 500 fold with 1x Assay Diuent For example Briefly spin the vial Item D 1 and pipette up and down to mix gently Add 10 ul of HRP conjugated anti rabbit IgG concentrate into a tube with 5 ml Ix AssayDiluent to prepare a 500 fold diluted HRP conjugated anti rabbit IgG solution 7 Cell Lysate Buffer should be diluted 2 fold with deionized or distilled water before use recommend to add protease and phosphatase inhibitors VII ASSAY PROCEDURE 1 Bring all reagents to room
4. temperature 18 25 C before use It is recommended that all samples or Positive Control should be run at least in duplicate Add 100 ul of each sample or positive control into appropriate wells Cover well with plate holder and incubate for 2 5 hours at room temperature or over night at 4 C with shaking 2 Discard the solution and wash 4 times with 1x Wash Solution Wash by filling each well with Wash Buffer 300 ul using a multi channel pipette or autowasher Complete removal of liquid at each step is essential to good performance After the last wash remove any remaining Wash Buffer by aspirating or 7 RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol decanting Invert the plate and blot it against clean paper towels 3 Add 100 ul of prepared 1x rabbit anti phospho Akt Ser473 antibody or 1x rabbit anti pan Akt Reagent Preparation step 5 to appropriate wells Incubate for 1 hour at room temperature with shaking 4 Discard the solution Repeat the wash as in step 3 5 Add 100 ul of prepared 1X HRP conjugated anti rabbit IgG to corresponding well Incubate for I hour at room temperature with shaking 6 Discard the solution Repeat the wash as in step 3 7 Add 100 ul of TMB One Step Substrate Reagent Item H to each well Incubate for 30 minutes at room temperature in the dark with shaking 8 Add 50 ul of Stop Solution Item I to each well Read at 450 nm immediately VIII ASSAY PROCEDURE SUMMARY
5. A Kit Protocol
6. RayBio Phospho Akt Ser473 and Pan Akt ELISA Kit For Measuring Phospho Akt Ser473 and Pan Akt in Human Mouse and Rat Cell Lysates User Manual Revised Mar 1 2012 RayBio Phospho Akt Ser473 and Pan Akt ELISA Kit Protocol Cat PEL Akt S473 T We Provide You With Excellent Protein Array System And Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 770 206 2393 Web www raybiotech com Email info Qraybiotech com RayBiotech Inc RayBio Phospho Akt Ser473 and Pan Akt ELISA Kit Protocol TABLE OF CONTENTS I JnttoOduCUOBo se ois o Eoda eue da rao EE RPOERE S 2 H Material Proyided iios sana ety et ats 3 MI Ne 4 IV Additional Materials Required 4 V Sample PrepatatiOEi isse eoe Eoo Ras 4 VI Reagent Preparation ccc cece cence ee eens 5 VIL Assay Procedute i e eoe ne 7 VIII Assay Procedure Summary 8 IX Tr ulver 9 i Positive Controle reaches 9 ii Recombinant Human PDGF Stimulation of NIH3T3 Cell LTH68 redes iue t wu ERRa Inca 10 SSN Ad 11 X ARCTCTENCCS en iehress ci i P Hoe E LEES RR RURVE DUE Ae Ta Fen lupis 12 XI Troubleshooting Guide sssuuuus 13 1 RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol I INTRODUCTION RayBio amp Phospho Akt Ser473 and Pan Akt ELISA Enzyme Linked Immunosorbent Assay kit is a very rapid convenient and sensitive assay kit that can monitor the acti
7. entrate 20x Item B 25 ml of 20x concentrated solution Assay Diluent Item E2 15 ml of 5x concentrated buffer For diluting cell lysate sample detection antibody Item C 1 and Item C 2 and secondary antibody Item D 1 concentrate Detection Antibody Akt Ser473 Item C 1 1 vial of rabbit anti phospho Akt Ser473 1 vial is enough to assay half microplate Detection Antibody Akt Item C 2 1 vial of rabbit anti pan Akt 1 vial is enough to assay half microplate HRP conjugated Anti rabbit IgG Item D 1 25 ul of 500x HRP conjugated Anti rabbit IgG concentrate TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffered solution Stop Solution Item I 8 ml of 0 2 M sulfuric acid Cell Lysate Buffer Item J 5 ml 2x cell lysis buffer not including protease and phosphatase inhibitors Positive Control A431S003 1 Item K 1 vial of lyophilized powder from A431 cell lysate HI STORAGE Upon receipt the kit should be stored at 20 C Please use within 6 months from the date of shipment After initial use Wash Buffer Concentrate Item B Assay Diluent Item E2 TMB One Step Substrate Reagent Item H Stop Solution Item I and Cell Lysate 3 RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol Buffer Item J should be stored at 4 C to avoid repeated freeze thaw cycles Return unused wells to the pouch containing desiccant
8. pack reseal along entire edge and store at 20 C Item D 1 store at 2 8 C for up to one month store at 20 C for up to 6 months avoid repeated freeze thaw cycles Reconstituted Positive Control Item K should be stored at 70 C IV ADDITIONAL MATERIALS REQUIRED Microplate reader capable of measuring absorbance at 450 nm Protease and Phosphatase inhibitors Shaker 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 Distilled or deionized water Tubes to prepare sample dilutions oo N DANA WN V SAMPLE PREPARATION Cell lysates Rinse cells with PBS making sure to remove any remaining PBS before adding the Cell Lysate Buffer Solubilize cells at 4 x 10 cells ml in 1x Cell Lysate Buffer we recommend adding protease and phosphatase inhibitors to Cell Lysate Buffer prior to sample preparation Pipette up and down to resuspend and incubate the lysates with shaking at 2 8 C for 30 minutes Microcentrifuge at 13 000 rpm for 10 minutes at 2 8 C and transfer the supernates into a clean test tube Lysates should be used immediately or aliquoted and stored at 70 C Avoid repeated 4 RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol freeze thaw cycles Thawed lysates should be kept on ice prior to use For the initial experiment we recommend to do a serial dilution testing such as 5 fold and 50 fold dil
9. uantitative measurement of multiple protein levels Protein Array ELISA Cell Based Phosphorylation ELISA Tissue MicroArray Protein Cytokine Chemokine Adiplokine Angiogenic factor Virus bacteria and infectious disease protein hormone Enzyme other Peptide Antibody Cytokine Adipokine Angiogenic factor Signal transduction Transcription factor Receptor Adhesion molecule Virus bacteria and other infectious agents Secondary antibody Tag antibody Immunoglobulin Hormone Cell surface Protease other Antibody array Protein array Peptide array ELISA Phosphorylation assay Tissue array 14 RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol Assay service just simply send your samples and get data in 1 to 2 weeks Antibody array Protein array ELISA Quantibody array Antibody production highest quality with very competitive price Monoclonal antibody Recombinant antibody Polyclonal antibody Phase display Antibody angineering Antibody conjugation Recombinant protein production Assay development Array printing Contact and non contact arrayers All kinds of substrates of your choice including glass slides membranes and plates 15 RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol Note 16 RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol This product is for research use only m mm munn Wr i 02004 RayBiotech Inc 17 RayBio Phospho Akt Ser473 and pan Akt ELIS
10. ution for your cell lysates with Assay Diluent Item E2 before use Note The fold dilution of sample used depends on the abundance of phosphorylated proteins and should be determined empiricallys More of the sample can be used if signals are too weak If signals are too strong the sample can be diluted further Cell Lysate Buffer should be diluted 2 fold with deionized or distilled water before use recommend to add protease and phosphatase inhibitors VI REAGENT PREPARATION 1 Bring all reagents and samples to room temperature 18 25 C before use 2 Item E2 Assay Diluent should be diluted 5 fold with deionized or distilled water before use 3 Preparation of Positive Control Briefly spin the Positive Control vial of Item K Add 500 ul Ix Assay Diluent Item E2 Assay Diluent should be diluted 5 fold with deionized or distilled water before use into Item K vial to prepare Positive Control 1 Solution See i Positive Control of part IX TYPICAL DATA 5 RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol for a typical result in page 9 Dissolve the powder thoroughly by a gentle mix it can be removed by centrifuge if any precipitate in the solution is found Pipette 300 ul 1x Assay Diluent into each tube Use the Positive Control 1 to produce a dilution series shown below Mix each tube thoroughly before the next transfer Ix Assay Diluent serves as the background Positive Control powder 500 ul 1x
11. vation or function of important biological pathways in cell lysates By determining phosphorylated Akt protein in your experimental model system you can verify pathway activation in your cell lysates You can simultaneously measure numerous different cell lysates without spending excess time and effort in performing a Western Blot analysis This Sandwich ELISA kit is an in vitro enzyme linked immunosorbent assay for the measurement of phospho Akt Ser473 and pan Akt lin human mouse and rat cell lysates help normalize the results of phospho Akt from different cell lysate being compared An pan Akt antibody has been coated onto a 96 well plate Samples are pipetted into the wells and Akt present in a sample is bound to the wells by the immobilized antibody The wells are washed and anti phospho Akt Ser473 or anti pan Akt is used to detect phosphorylated or pan Akt After washing away unbound antibody HRP conjugated anti rabbit IgG is pipetted to the wells The wells are again washed a TMB substrate solution is added to the wells and color develops in proportion to the amount of Akt Ser473 or pan Akt bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm 2 RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol II MATERIAL PROVIDED 1 2 oo 10 Akt Microplate Item A 96 wells 12 strips x 8 wells coated with anti pan Akt antibody Wash Buffer Conc
12. wash b Make fresh wash buffer buffer 4 Positive Control a Improper storage of the a Upon receipt the kit Low signal ELISA kit should be stored at 20 C Store the positive control at 70 C after reconstitution b Stop solution b Stop solution should be added to each well before measurement and read OD immediately c Improper primary or secondary antibody c Ensure correct dilution 13 RayBio Phospho Akt Ser473 and pan Akt ELISA Kit Protocol RayBio ELISA kits Over 200 ELISA kits custom ELISA kit choose from over 500 list visit www raybiotech com for details RayBiotech Inc the protein array pioneer company strives to research and develop new products to meet demands of the biomedical community RayBio s patent pending technology allows detection of over 180 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 Phosphotyrosine kits Recombinant Proteins Antibodies and custom services Antibody Array Cytokine Antibody Array Simultaneous detection up to 200 proteins cytokine chemokine growth factor adipokine angiogenic factor protease in one experiment Phosphorylation Antibody Array e RTK antibody array e EGFR phosphorylation antibody arrays Label based antibody array Simultaneous detection more than 500 proteins in one experiment Quantibody Array Q
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