Anti-GAPDH antibody (HRP) - Loading Control (ab9385)

Dear Sir/Madam,
My quesions regarding ab9385 anti-GAPDH antibody HRP conjugated.
Western blot conditions:

5% BSA for 1h at room temperature
ECL
dilution used 1:5000 my PVDF membrane stored at 4 degree in PBS-0.05 % tween 20 for two weeks notice that this membrane was blocked with milk before storage
I blocked with 5% BSA as mensioned in your
direction that (milk cause no band problem) then I did wash 4 time
with PBS-0.05 % tween 20 10 min each then I dilute the GAPDH- HRP
loading control 1:5000 in 5% BSA-PBS-0.05 % tween 20 and I incubated
overnight in fridge 4 degree then I did wash for 4 times each 10 min
then I added secondry antibody for biotinlyted ladder diluted 1:1000
in 5% BSA-PBS-0.05 % tween 20 for 1h then I wahed it 4 times with
PBS-0.05 % tween 20 10 min each then exposed to ECL for 90 min then
expose 1 min
however I got high background and not clear band my cells is HUVEC
have you tried this loading control in this cell? and what do you
think the problem ? I attached to you the image .. Hopes I explained my prolem clearly please do not hesitate to
contact me if you need further information.
I look forward for your reply.
Kind regards;

ANSWER:

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

In order to better understand the problem, I would like to send you a questionnaire with some additional questions about the protocol used, as well as the order details. All this information is crucial to help us understand the possible causes of the problem.

Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results.

I look forward to receiving your reply. Please do not hesitate to contact us if you need any more advice or information.

dear Abcam,

I am searching for a house keeping antibody for Candida albicans. i have already tried 2 of them: PDI and CDC-42 from another company which were not sucessfull. What you recommend? I need an answer as soon as possible. Thanks a lot

ANSWER:

Thank you for your enquiry and your interest in our product.

We do not test our antibodies in Candida albicans on a regular basis, so it is not known if any of them would work in this species. However, the most popular loading controls in mammalian experimental system are beta actin, GAPDH, alpha tubulin.

ab8224:Anti-beta Actin antibody [mAbcam 8224] - Loading Control

- tested in:Mouse, Rat, Rabbit, Chicken, Cow, Cat, Dog, Human, Pig, Saccharomyces cerevisiae, Xenopus laevis, Fruit fly (Drosophila melanogaster), Schizosaccharomyces pombe, Chinese Hamster, Other

ab9385:Anti-GAPDH antibody (HRP) - Loading Control

- tested in: Mouse, Rat, Human, Saccharomyces cerevisiae, Xenopus laevis



If you need any further assistance in the future, please do not hesitate to contact me.

I'm using this antibody in WB with e. Coli lysate as a control. I'm seeing a strong band at about 30 kDa and nothing at 38 kDa. Also the band strength seems to be varying with my treatment.

How was this antibody raised? Was there every any expression in e. Coli?

ANSWER:

Thank you for your inquiry.

I heard back from the lab that the immunogen for ab9385 is a recombinant protein and rabbits were immunized with this recombinant sourced from Sigma (G6019). As this was not performed in-house we have no details about how the antigen was generated, but if this information may affect your assessment of your data you may consider contacting Sigma for the process/protocol information.

http://www.sigmaaldrich.com/catalog/ProductDetail.do?D7=0&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&N4=G6019%7CSIGMA&N25=0&QS=ON&F=SPEC

I’ve looked through our historical WBs and also the WB Abreviews we have, but I was not able to find any data for testing against E. coli samples for your comparison.

I hope this information is helpful. Please contact us with any other questions.

Hi, Blocking with BSA worked. I'll need to do some optimization, but I now have bands to work with so it should be straightforward. Thank you for your help!

ANSWER:

Thank you for letting me know the result of the troubleshooting tip. I am glad to hear that changing to BSA blocking solution helped to overcome the initial 'no bands' with milk blocking solution.

If there is anything else that I can help you with, please do not hesitate to contact me.

Good luck in your experiment!

Thank you for your reply.

I attached the representative blots in this email, showing the samples are in good condition as I can detect other target bands (Rhodamine labeled fibronectin about 250kDa) perfectly. (In Blot 1) But in this blot I am using the ab9485, which doesn't work at all, and that's why your company send me a 9385 to try.

The second blot is kind of complicated. I cut the membrane into three pieces. You can focus on the piece in the right side, with 1st ab HRP-rabbit-GAPDH and 2nd ab X on top of it. I did get a band, but it is so faint and it is a 10 min exposure (I usually only need 1min exposure). And this one is using 5%milk and 1%BSA as blocking agent.

The third blot is my last try using 3%BSA as blocking agent. The signal is strong but also have a high background. And the detected protein has a size around 17kDa, which is really wierd.

I apologize for my poor English. I hope I have explained clearly enough. If you need more information, you can reply my email or call me.

Thank you for your help

ANSWER:

Thank you for getting back to me with your blot images.

We have recently tested this antibody in our lab and the antibody gave very good results. I am interested in the results that you have been obtaining. It appears that you have been able to detect a Rhodamine tagged anti- Fibronectin antibody with relative ease suggesting that the problem that you have been finding is not related to the samples that you have been using. However, can you tell me whether the experiments that you performed to detect fibronectin was performed in parallel with the experiments to detect GAPDH; were they from the same sample preparation?

With regards the 17KDa protein that you have been detecting using anti-GAPDH; this is certainly not related to GAPDH. However, I was particularly interested in the central blot that you emailed me. Can you tell me how this was generated as this produced a strong band of approximately the correct size. Were you amplifying the signal using an additional HRP conjugated secondary?

I appreciate your comments and look forward to hearing from you.