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We probed human placenta samples (60 ug) for the efflux transporter BCRP using your BCRP/ABCG2 primary antibody (ab63907, lot no: GR42347) for the first time. Our positive controls were mouse kidney lysates and human embryonic kidney (HEK) cells that overexpress the human BCRP. As a negative control we used the HEK cells that were overexpressing an empty vector. Additionally we added the same primary anitbody and the competing peptide (ab100820, lot no: GR53148) to another blot with the identical samples to be sure that the antibody is specific for BCRP. The positive controls (mouse kidney) did not show up, EV cells showed BCRP expression, and the competing peptide did not eliminate the bands. Below are some of the experimental conditions Blocking conditions: 5% milk in PBS/0.5% Tween, 1 hr Detection method: Chemiluminescence 20 minutes Electrophoresis: 160 V, 120 mA, 25 W 1.5 hours in 1x NuPage MOPS SDS Running Buffer (Life Technologies) Transfer: 7 minute dry transfer (Life Technologies) Primary ab: 1:1000 in 2% milk PBS/T overnight 4C and Primary + competing peptide (1:1000) Wash: 3x10 min PBS/T Secondary ab: 1:5000 in 2% milk PBS/T 1 hr RT Wash: 3x10 min PBS/T See powerpoint slide for images of blots. Thank you for your time.
Asked on Jan 22 2013
Thank you for taking time to contact us. I am sorry to hear that this antibody is not providing satisfactory results.
The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.
Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab63907 and ab100820. I would also appreciate if you can confirm some further details to help us better understand the problem with the antibody.
We haven't tested this antibody using mouse samples (only human and pig), so we are unsure how or if this antibody will work with mouse samples. We predict that the antibody may bind due to high sequence homology (>90%), but these are the first testing data we've seen so it may be that the antibody is not able to bind the mouse protein.
How were the samples prepared? For multiplass membrane proteins, we would recommend using RIPA buffer and heating to 70C for 5 - 10 mins rather than boiling since boiling can cause aggregates.
What percentage gel are you using? Also for the transfer, you may want to choose a wet transfer for these types of proteins. Make sure the transfer buffer has 0.1% SDS in it since this protein is hydrophobic and may have trouble entering the gel. This may explain why you are seeing such "wispy" bands in the blot.
What MW is that main band running at? Is it at the expected 72 kDa?
In order to use a blocking peptide correctly, the peptide needs to be incubated with the antibody in 5 - 10 times molar excess. Using both the antibody and the peptide at the same concentration will not allow proper blocking. The blocking assay looked like it worked though for the HEK lanes in the WB. Have you confirmed the empty vector since this band is blocked and is therefore considered specific?
Should the suggestions not improve the results, please do let me know.
In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.
I hope this information is helpful, and I thank you for your cooperation.
Answered on Jan 22 2013