Question (20830) | Anti-Cytochrome P450 1A1 + 1A2 antibody (ab37131)

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Question

Hi, I want ues your products ab 2778 and ab37131 in ELISA method. Please inform me how can i prepare a standard curve for the concerned proteins to quantify them? Do you also have the standard proteins concerned with the above antibodies? Please let me know at your earliest.

Answer

I have been informed by the suppliers for ab37131 and ab2778 that we do not supply the standard proteins unfortunately. For ab2778, anti-Nonspecific Cytotoxic Cell antibody, the lab team has informed me that this antibody was actually tested in ELISA by a customer. The only information we have available is that the researcher used a peptide capture ELISA to determine the active site for the receptor protein NCCRP-1. The lab has suggested that if one wants to quantify NCCRP-1, recombinant peptides would have to be used. Additionally, please keep in mind that this antibody is an IgM isotype. If you experience problems with this product please do contact us, as we have a guarantee on the quality of the antibody in Rat, Catfish, Cow, Dog, Horse, Pig, and Tilapia in ELISA. For ab37131, as I mentioned we don´t have a Rainbow trout CYP1A protein standards. However, I was able to find the specific protocol to be used for ELISA, which I have added to our datasheet and also copied below for your convenience. BUFFERS/REAGENTS 1. Coating buffer:50 mM carbonate-bicarbonate, pH 9.6. 2. PBS:Phosphate buffered saline, pH 7.3 3. Washing buffer: PBS, 0.05% Tween-20 4. Blocking/Dilution buffer: 1% BSA in PBS. 5. Substrate solution. I. ASSAY PROCEDURE Day 1: Coating with samples and positive control Frozen samples and positive control should be thawed on ice. 1. Dilute samples in Coating buffer: Please note: The optimum coating concentration must be determined for each assay, but these dilutions/concentrations can be generally recommended: Liver microsomes: 10 µg/ml Post-mitochondrial supernatants (PMS): 40 µg/ml Plasma samples: 1:1000 Purified proteins: 5-10 µg/ml 2. Dilute the positive control in coating buffer to reach desired concentration. 3. Add 100 µl coating buffer to each of two wells (duplicate analysis) or three wells (triplicate analysis). These wells will be used to determine the Non- Specific Background signal (NSB). 4. Add 100 µl diluted positive control to each of two or three wells. 5. For each sample, add 100 µl to each of two or three wells. 6. Incubate at 4 °C overnight Please note: Coating for 1 hour at room temperature or 37ºC is possible, but this may alter the sensitivity of the assay depending on the nature of the sample. Day 2: Blocking the wells 7. Wash the wells 3 times with 300 µl Washing buffer per well. 8. Add 200 µl of the Blocking/Dilution buffer to each well. Incubate at room temperature for 30-60 min. Incubation with Primary antibody 9. Empty the wells. 10. Dilute the Primary antibody in Blocking/Dilution buffer. Add 100 µl of the antibody solution to each well. Incubate at 37 °C for 1 hour. Please note: Incubation with the Primary antibody may be performed for 2 hours at 37 °C or at 4°C overnight. Depending on the Primary antibody, this may alter the sensitivity of the assay. Incubation with Secondary antibody 11. Wash the wells 3 times with 300 µl Washing buffer per well. 12. Dilute the Secondary antibody in Blocking/Dilution buffer for each plate run in the assay. Add 100 µl of the antibody solution to each well. Incubate at room temperature for 1 hour. Please note: Increasing the incubation time of the Secondary antibody to 2 hours or incubating at 37ºC may increase assay sensitivity. Development of the plate Please note: The Substrate solution should be prepared just before proceeding to the next step. 13. Wash 5 times with 300 µl Washing buffer per well. 14. Add 100 µl substrate solution to each well. Incubate at room temperature for 15 min Please note: If the reaction is weak, the incubation time may be extended up to 30 min. 15. Stop the reaction by adding 50 µl 2M H2SO4 to each well. 16. Read the absorbance at 492 nm. I hope this information will help you, good luck with your research.

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