Rat VEGF R-2 ELISA Kit Manual
Contents
1. 96 well dish Do not use the ELISA wells directly to perform the dilutions there should be 6 wells in duplicate The final sample volume in each well should be 50uIl Table 2 Standard dilutions Displayed below for rat mouse and human use a similar dilution protocol 1800 pg ml Standard No 6 100l Standard peptide 50ul standard diluent mix Remove 100ul to make Standard No 5 1200 pg ml Standard No 5 100ul Standard No 6 50ul standard diluent mix Remove 100u to make Standard No 4 600 pg ml Standard No 4 100ul Standard No 5 100ul Standard diluent mix Remove 50ul to make Standard No 3 Remove and discard 100ul 300 pg ml Standard No 3 50ul Standard No 4 50ul Standard diluent mix Remove 50ul to make Standard No 2 150 pg ml Standard No 2 50ul Standard No 3 50ul Standard diluent mix Remove 50ul to make Standard No 1 75 pg ml Standard No 1 50ml Standard No 2 50ml Standard diluents mix Remove and discard 50ul 2 Set up 2 blank wells separately In blank wells add 40ul sample diluent and 10ul solution used to solubilize the samples i e vehicle do not add sample Perform all other procedures of the assay except do not add HRP conjugated antibody to the blank wells 3 For wells with experimental samples add 40ul sample diluent in each well then add 10ul of sample diluted in sample diluent This creates a 5X dilution factor of sample which should be account2. absorbance multiplate reader is employed to measure OD at 450 nm Correlation of absorbance in the assay samples with the peptide standard provided at known concentration is used to determine p65 concentration in the assay samples Standards concentrations and assay ranges Species Standard Peptide Concentration Assay Range Human 2250 pg ml 100 2000 pg ml Mouse 1800 pg ml 50 1500 pg ml Rat 2700 pg ml 30 1800 pg ml Table 1 Provided Materials in the Kit Materials provided with the kit 96 determinations Wash solution 30X Materials required but not supplied 1 37 C incubator 2 Standard microplate reader 3 Distilled water FIVEphoton Biochemicals 4907 Morena Blvd Ste 1403 San Diego CA 92117 Tel 800 462 4507 Fax 858 345 5291 Page 2 of 6 iit FIVEphoton Biochemicals www fivephoton com 4 Disposable tubes for sample dilution 5 Absorbent paper Important notes and preparation for the assay 1 It is recommended that the experimenter perform preliminary tests using reagents of this kit to identify the sample dilution required to meet the assay range Perform a preliminary assay with samples using standards at the high and low dilution Suspend and dilute experimental samples into the sample diluent provided with this kit to meet the requirements of the assay range Adilution series of several samples may be required to identify the correct concentration that meets th
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4. e assay range Concentrate or dilute test samples if adjustment is needed Set aside sufficient experimental sample for reserve to repeat the assay 2 Determine whether the sample vehicle reacts with the assay Dilute the sample in sample diluent or prepare samples in another vehicle to prevent inadvertent experimental readings 3 The kit should be equilibrated to room temperate for 30 min prior to performing the assay Store opened microelisa plates in a sealed plastic bag at 4 C A multi channel sampler that has been calibrated for accuracy is a preferred method to apply samples Plates should be sealed during the assay Wells should not be allowed to dry 4 Perform dilution of the standards in separate tubes or 96 well plates not in the ELISA wells Transfer solutions simultaneously to the ELISA dish It is recommended that samples are assayed in duplicate to address pipetting error Use new applicator tips and ELISA plate sealants to avoid cross contamination Do not mix reagents from other ELISA kits Note that sodium azide in samples that is not washed away may inhibit HRP When calculating concentration of your sample from the assay make sure to take into account the dilution factor 3 Pon o gi 0 If wash solution crystallizes during storage at 4 C heat solution at 37 C and shake until crystals suspend Sample preparation for p65 RelA ELISA assay Using ELISA to measure NF B activation p65 RelA is localized in cells in th
5. e cytosol and in the nucleus It translocates from the cytosol to the nucleus when NF xB is activated To isolate a total cellular p65 RelA pool for ELISA assay use a non denaturing detergent lysis buffer such as the Fivephoton Biochemicals ELISA Lysis Buffer Part ELSP 1 The protocol associated with the lysis buffer can be used to assay the total p65 cellular pool This ELISA assay can also be used to measure NF xB activation by partitioning a cytoplasmic and nuclear p65 pool and assaying the concentration of p65 in the respective cellular compartment The ratio of nuclear relative to cytoplasmic p65 provides an index of NF B activation The Fivephoton Biochemicals Nuclear Protein Isolation Kit Part NPI 1 or similar and the associated protocol can be employed to isolate nuclear and cytoplasmic fractions for ELISA Assay procedures Standard and Sample Preparation Standards samples and blanks should be added simultaneously to wells Prepare the standards samples and blanks in a separate 96 well dish and transfer simultaneously to the ELISA dish FIVEphoton Biochemicals 4907 Morena Blvd Ste 1403 San Diego CA 92117 Tel 800 462 4507 Fax 858 345 5291 Page 3 of 6 mii FIVEphoton Biochemicals www fivephoton com Assay procedure 1 Set aside and mark 12 wells for standard peptide dilutions Configure six concentrations of standard peptide in duplicate suspended and mixed as indicated below in separate tubes or in a separate
6. ed for later when calculating sample concentration 4 Transfer the Standard Solutions Blank Well Solutions and Experimental Samples simultaneously to the ELISA dish from the 96 well dish used for solution preparation 5 Use the closure membrane or a zip lock bag to enclose the plate mix gently with a rotating table and incubate for 30 min at 37 C or 1 hr at RT 6 Dilute the 30X Wash Solution with dH20 Make 3 ml of diluted wash solution for each assay well 7 After the first incubation period discard the liquid in the wells by gently aspirating Turn the plate upside down and gently pat dry the plate with an absorbent paper To wash the wells fill each well with 100 ul of FIVEphoton Biochemicals 4907 Morena Blvd Ste 1403 San Diego CA 92117 Tel 800 462 4507 Fax 858 345 5291 Page 4 of 6 mii FIVEphoton Biochemicals www fivephoton com 10 11 12 13 diluted Wash Solution oscillate gently with the rocker table for 30 sec and then aspirate off liquid Pat dry the microplate with absorbent paper Repeat the wash steps 5 times Simultaneously transfer 50 ul HRP conjugate reagent to each well except for blank wells Rotate gently at 37 C for 30 min or R Wash the wells as described in above 5 times Remove liquid from the wells Transfer 50 ul of Chromogen Solution A and then 50ul of Chromogen Solution B to each well prepare these solutions beforehand in a separate 96 well dish Gently mix for 15
7. mii FIVEphoton Biochemicals www fivephoton com NF kappa B RelA p65 Sandwich Elisa FIVEphoton General Protocol Biochemicals For research use only Not for diagnostics Use the protocol shipped with your kit for your experiments FIVEphoton Biochemicals 4907 Morena Blvd Ste 1403 San Diego CA 92117 Tel 800 462 4507 Fax 858 345 5291 Website www fivephoton com customersupport fivephoton com Store 4 C Expires 6 month after arrival Safety Stop solution contains acid Avoid all contact and inhalation 1 Copyright FIVEphoton Biochemicals 2013 iit FIV Ephoton Biochemicals BACKGROUND This ELISA kit targets the RelA p65 subunit of NF kappa B using a 96 well strip plate coated with affinity purified www fivephoton com antibody specific for p65 together with a provided p65 peptide to calibrate absorbance to p65 concentration A horse radish peroxidase HRP conjugated polyclonal antibody and chromogens are employed for colorimetric detection Simultaneous addition of a dilution series of standard peptide as well as assay samples are applied to individual wells Following a 30 min 1 hr incubation period unbound material is washed and the HRP conjugated antibody is added to wells After an additional 30 min 1 hr incubation unbound antibody is rinsed and chromogen solutions are added to the wells The reaction between chromogens and HRP is allowed to proceed for 15 min prior to stopping An
8. min at 37 C in the dark Simultaneously add 50 ul of Stop Solution to each well Upon addition of stop solution the blue color should immediately change to yellow Measure the optimal density OD at 450 nm within 15 minutes of adding the stop solution Set the blank wells as zero If sample readings are higher than the most concentrated standard you may wish to dilute the samples and assay again Data Analysis 1 Compile a standard curve using the blank standard solutions and the corresponding OD values You may wish to calculate a linear regression equation from the standard curve to determine the concentration of your samples Remember that samples were diluted 5 fold in the assay in your final calculation Other forms of analysis to calculate concentrations of your samples can also be applied Flow chart of the procedures Prepare standards blank and samples Add samples to wells incubate for 1 hr at RT or 30 min at 37 C Wash each well five times Add HRP conjugate antibody to each well incubate for 1 hr at RT or 30 min at 37 C Wash each well five times FIVEphoton Biochemicals 4907 Morena Blvd Ste 1403 San Diego CA 92117 Tel 800 462 4507 Fax 858 345 5291 Page 5 of 6 wii FIVEphoton Biochemicals www fivephoton com Add chromogen solutions A and B 15 min at 37 C JL Add stop solution JL Measure OD 450 nm within 15 min FIVEphoton Biochemicals 4
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