Home

Manual

1. Add 100 ul of prepared 1x HRP conjugated anti rabbit IgG against anti AKT anti AKT S473 anti GSK3p anti GSK3p S9 and anti mTOR S2448 or 1x HRP conjugated Streptavidin against biotinylated anti mTOR see Reagent Preparation step 6 to corresponding wells Incubate for 1 hour at room temperature with shaking 7 Discard the solution Repeat the wash as in step 3 8 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 9 Add 50 ul of Stop Solution Item I to each well Read at 450 nm immediately RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol VIII ASSAY PROCEDURE SUMMARY 1 Prepare all reagents samples and standards as instructed 2 Add 100 ul sample or positive control to each well Incubate 2 5 hours at room temperature or overnight at 4 C 3 Add 100 ul prepared primary antibody to each well Incubate 1 0 hours at room temperature J 4 Add 100 ul prepared 1X HRP Conjugated antibody solution Incubate 1 hour at room temperature 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 RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol IX TYPICAL DATA ELISA data analysis Average the duplicate readings for each sample or positive i Positive Control NIH 3T3 cells were treated wit
2. 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 freeze thaw cycles Thawed lysates should be kept on ice prior to use For the initial experiment we recommend a serial dilution such as 5 fold to 50 fold for your cell lysates with Assay Diluent Item E2 before use RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol Note The fold dilution of sample used depends on the abundance of phosphorylated proteins and should be determined empirically 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 Assay Diluent Item E2 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 450 ul 1x Assay Diluent Item E2 into Item K to prepare Positive Control P 1 solution Dissolve the powder thoroughly by a gentle mix it can be removed by centrifuge if any precipitate in the solution is found Pip
3. rat and a variety of other species Visit www raybiotech com for the complete list 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 400 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 15 RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol This product is for research use only 2004 RayBiotech Inc 16 RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol
4. with 5 0 mL Ix Assay Diluent pipette up and down to mix gently to prepare a 1000 fold diluted HRP conjugated anti rabbit IgG solution Mix well 7 Cell Lysate Buffer should be diluted 2 fold with deionized or distilled water before use recommend to add protease and phosphatase inhibitors RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol VII ASSAY PROCEDURE 1 Bring all reagents to room temperature 18 25 C before use It is recommended that all samples or Positive Control should be run at least in duplicate 2 Add 100 ul of each sample 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 96 well microplate coated with pan antibodies Anti AKT Anti GSK3b Anti mTOR 123 4 5 6 7 8 9 10 11 12 Ionmuoou r 3 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 decanting Invert the plate and blot it against clean paper towels 4 Add 100 ul of prepared 1x detection antibody Reagent Preparation step 5 to appropriate wells Incubate for 1 hour at room temperature with shaking RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol 5 Discard the solution Repeat the wash as in step 3 6
5. 1 1 Item K 1 vial of lyophilized powder from NIH3T3 cell lysate RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol IIl 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 Buffer Item J should be stored at 4 C to avoid repeated freeze thaw cycles Return unused wells to the pouch containing desiccant pack reseal along entire edge and store at 20 C Item D 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 219g tn WN RK V SAMPLE PREPARATION Cell lysates Rinse cells with PBS making sure to remove any remaining PBS before adding the lysis buffer Solubilize cells at 4 x 10 cells ml in 1x Lysis Buffer we recommend adding protease and phosphatase inhibitors to lysis buffer prior to sample preparation Pipette up
6. GSK3p column 5 8 and mTOR column 9 12 antibodies Wash Buffer Concentrate 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 and HRP conjugated IgG Concentrate Item D Detection Antibody Akt Ser473 Item C 1 1 vial of rabbit anti phospho Akt Ser473 1 vial is enough to assay 3 strips Detection Antibody Akt Item C 2 1 vial of rabbit anti Akt 1 vial is enough to assay 3 strips Detection Antibody GSK3p S9 Item C 3 1 vial of rabbit anti GSK3b S9 1 vial is enough to assay 3 strips Detection Antibody GSK3p Item C 4 1 vial of rabbit anti GSK3p 1 vial is enough to assay 3 strips Detection Antibody mTOR S2448 Item C 5 1 vial of rabbit anti mTOR S2448 1 vial is enough to assay 3 strips Detection Antibody mTOR Item C 6 1 vial of biotinylated anti mTOR 1 vial is enough to assay 3 strips HRP conjugated Anti rabbit IgG Item D 1 25 ul of 1000x concentrated HRP conjugated anti rabbit IgG HRP conjugated Streptavidin Item G 25 ul of 300x concentrated HRP conjugated Streptavidin 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 3T3S00
7. RayBio Phospho A KT GSK38 mTor ELISA Kit For measuring phosphorylated AKT S473 GSK3p S9 mTOR 2448 and Total AKT GSK3p mTOR in human cell lysates User Manual Revised Aug 15 2014 RayBio Phospho AK T GSK30 mTor ELISA Kit Protocol Cats PEL AKT SK asa mmm m m jm mm mmm RayBiotech Inc ISO 13485 amp GLP Certified Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 770 206 2393 Web www raybiotech com Email info raybiotech com RayBiotech Inc RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol TABLE OF CONTENTS CPPCC MODs entree ocounenetsceccsewgacarsnceaceses 2 IH Material Provided ios 3 MI SSIODUBE x nctcsien ete da enbasasictivae seta eaneneoiateineds 4 IV Additional Materials Required 4 V Sample Preparation c cece eee eee eee eee 4 VI Reagent Preparation cce cece eee ee eens 5 VII Assay Procedute a iscsi ee exco ees Quen eer exe 7 VIII Assay Procedure Summary 9 IX Typical Daas ote osccsocsnseiedreteseece versie eins 10 i Positive Control ii ose cete ker Ea etra ien 10 ii PDGFBB Stimulation of NIH3T3 COLLE IOS soin eetobetdantont iO IDEE 11 ii EGF Stimulation of A431 Cell Lines 13 X Troubleshooting Guide ssusesus 14 RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol y Pp I INTRODUCTION RayBio Phospho AKT GSK3f mTor ELISA Kit is a very rapid c
8. ette 300 ul 1x Assay Diluent into each tube Use the Positive Control P 1 solution to produce a dilution series shown below Mix each tube thoroughly before the next transfer 1x Assay Diluent serves as the background See i Positive Control of part IX TYPICAL DATA for a typical result on page 10 Positive Control Item 150u 150 4 Ss 150 ul K 450 ul lx A ate a l AN f Sv eV f 3958 RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol 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 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 detection antibody concentrate should be diluted 30 fold with 1x Assay Diluent and used in step 4 of Part VII Assay Procedure 6 Briefly spin the HRP conjugated anti rabbit or Streptavidin Item D 1or G before use HRP conjugated anti rabbit IgG concentrate should be diluted 1000 fold with 1x Assay Diluent or HRP conjugated Streptavidin concentrate should be diluted 300 fold with 1x Assay Diluent For example Briefly spin the vial Add 5 ul of HRP conjugated anti rabbit IgG concentrate into a tube
9. h recombinant human PDGFBB at 37 C for 10 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 Phospho AKT S473 Assay Diluent 1 5 OD 450 nm o P 1 P 2 P 3 P 4 P 0 Positive control dilution series Phospho GSK3b S9 Assay Diluent 2 5 OD 450 nm P 1 p 2 P3 P 4 P 5 Positive control dilution series 10 RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol ii Recombinant Human PDGFBB Stimulation of NIH 3T3 Cell Lines NIH 3T3 cells were treated or untreated with 50 ng ml recombinant human PDGFBB for 10 min Cell lysates were analyzed using this phosphoELISA and Western Blot A ELISA Analysis of Phospho AKT 3 0 25 mum NIH3T3 NIH3T3 PDGF E 2 0 c i Q 151 ll Q O 1 0 0 5 4 oi Phospho Akt Ser473 Pan Akt B Western Blot Analysis of Phospho AKT PDGFBB 0 10 0 10 Min Anti phospho Akt Anti pan Akt Ser473 RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol A ELISA Analysis of Phospho GSK3p 3 2 i Untreated 1 B PDGFBB 0 phospho GSK3b pan GSK3b OD 450 nm B Western Blot Analysis of Phospho GSK3p 0 10 0 10 Min PDGFBB Anti GSK3p S9 Anti pan GSK3f 12 RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol iii Recombinant Human EGF Stimulation of A431 Cell Lines A431 cells were treated or untreated
10. onvenient and sensitive assay kit that can monitor the activation or function of important biological pathways in human cell lysates By determining phosphorylated AKT GSK3p and mTOR 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 Blotting analysis This Sandwich ELISA kit is an in vitro enzyme linked immunosorbent assay for the measurement of human phospho AKT total AKT phospho GSK3 total GSK3p phospho mTOR and total mTOR For each target a capture antibody has been coated onto microwells Samples are pipetted into the wells and target protein present in a sample is bound to the wells by the immobilized antibody The wells are washed and a detection antibody is used to detect the captured target protein After washing away unbound antibody an HRP conjugated secondary antibody 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 target protein bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol II MATERIAL PROVIDED 1 10 11 I2 13 14 I5 Microplates Item A 96 wells 12 strips x 8 wells coated with anti pan AKT column 1 4
11. with 100 ng ml recombinant human EGF for 20 min Cell lysates were analyzed using this phosphoELISA and Western Blot A ELISA Analysis of Phospho mTOR 1 EJ Untreated B EGF 0 phospho mTOR pan mTOR OD 450 nm B Western Blot Analysis of Phospho mTOR 0 20 Min EGF Anti mTOR S2448 Anti pan mTOR 13 RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol X TROUBLESHOOTING GUIDE Problem Cause Solution 1 Sample signals a Too low a Sample concentration Increasing sample is too low concentration b Too high b Sample concentration Reducing sample is too high concentration 2 Large CV a Inaccurate pipetting Check pipettes 3 High background a Plate is insufficiently washed b Contaminated wash buffer Review the manual for proper washing If using an automated plate washer check that all ports are unobstructed Make fresh wash buffer 4 Low positive control signal a Improper storage of the ELISA kit b Stop solution c Improper primary or secondary antibody dilution Upon receipt the kit should be stored at 20 C Store the positive control at 70 C after reconstitution Stop solution should be added to each well before measurement and read OD immediately Ensure correct dilution 14 RayBio Phospho AKT GSK38 mTor ELISA Kit Protocol RayBio ELISA kits Choose from over 1 000 ELISA kits for human mouse

Manual

Contents

Download Pdf Manuals

Related Search

Related Contents