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I have already purchased goat polyclonal antibody to GPCR GPR40 and having a problem running a western blot. Currently, I am keep seeing a larger band instead of 28 kDa band, as it is shown on the data sheet. I have used .03 ug/ml concentraion of this anitbody and incubated it for an hour as it is suggested on the data sheet and seccondary anti-goat(1:6000 dilution) was added and incubated for an hour. I was wondering if it would be possible to obtain an exact protocol that was used in detecting a band that is shown on the data sheet of this antibody.
Asked on Aug 14 2006
Thank you for taking the time to contact us with your enquiry. I am very sorry you are having difficulty obtaining satisfactory results from this antibody (Ab32889) in western blotting. Below, I have copied a western blotting procedure used to test this antibody in the laboratory. I hope it will help. Bands of incorrect size are sometimes caused by inefficient denaturing and reducing of the protien. Your SDS (denaturing) sample buffer should contain a reducing agent (mercaptoethanol or DTT). This procedure mentions boiling for 5 minutes for denaturing and reducing, but you can try increasing this to 10 minutes if you have not already done so. Good luck with your experiments. If you have any more difficulty, please don't hesitate to get back to us. If you could please fill in the technical questionaire (see link below) and perhaps include and image of your blot, including a positive control and molecular weight markers, this would be very helpful. Thank you for your time. https://www.abcam.com/index.html?section=western&pageconfig=technical&mode=questionaire PROCEDURE: Tissue Lysis. Tissue chunks were weighed and cut into approx 1mm cubes using a razor blade. The tissue was transferred to a handheld homogenizer and 3 ml of ice-cold RIPA buffer was added per 1g of tissue. The tissue was gently homogenised over 20 minutes on ice. The resulting lysate was aliquotted into 1.5 ml microfuge tubes and centrifuged at 13,000 rpm for 5 min in a microfuge. The supernatant was transferred into clean tubes and its protein concentration was measured with protein assay. The concentration was then adjusted to 5 mg/ml with RIPA lysis buffer. An equal volume of 2 x SDS sample buffer was added and the cell lysate was boiled for 5 minutes. Lysates were stored at -80C until use.(RIPA buffer = 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 µg/ml Aprotinin, 5 µg/ml Leupeptin, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS). - SDS PAGE. Samples were run at 200V constant on a 12% acrylamide SDS-PAGE mini gel - using Biorad Mini-Protean 3 kit and protocols. Before loading samples had 5% (v/v) 2-ME added and were boiled for 3 minutes. - Transfer. We used a Biorad Mini Trans-Blot, constant 100 V for 1 hour. Transfer Buffer was 20 mM Tris pH 8.0, 150 mM Glycine, 10% Methanol. We transferred to PVDF membrane and stained with Ponceau Red to evaluate the transfer. - Staining. The membrane was blocked in 2.5% skimmed milk in TBS-T (TBS + 0.05% Tween-20) for 1 hr at room temperature with agitation. Primary antibody was incubated for 1 hr at room temperature with agitation. We used anti-goat-HRP secondary antibody for 1 hr at room temperature with agitation. We washed with TBST three times after primary and secondary antibody, each wash lasting for 5-10 mins. ECL-plus was used rather than ECL, which is considerably more sensitive. Final detection was on autoradiography film.
Answered on Aug 16 2006