Anti-ALDH1A1 antibody [EP1933Y] (ab52492)

What is the homology of the immunogen for this antibody against dog tissue?

ANSWER:

Thank you for contacting Abcam.



The immunogen shares 88% identity with canine Aldh1a1.

Please let me know if there is anything else I can help you with.

DESCRIPTION OF THE PROBLEM Clean band shows at 95kDa rather than 55kDa as shown on the data sheet. No other unspecific bands are seen. SAMPLE Human Breast cancer cell line and ovarian cancer cell line tested PRIMARY ANTIBODY Abcam, human, rabbit monoclonal (ep1933Y), in 50:50 PSB: Licor blocking buffer and 0.1% Tween. Used at 1/2000, 1/1000, 1/500 and 1/250. Incubated overnight at 4degrees. washed in PBS-T (0.1%) DETECTION METHOD Licor Odyssey infrared imaging system at 700nm POSITIVE AND NEGATIVE CONTROLS USED SKBR3 no negative used (please recommend) ANTIBODY STORAGE CONDITIONS -20degrees SAMPLE PREPARATION Lysate made in RIPA buffer with inhibitors: PMSF, sodium orthovanadate, leupeptin and aprotinin. AMOUNT OF PROTEIN LOADED 50ug ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE (reducing), 8% gel TRANSFER AND BLOCKING CONDITIONS tris-glycine transfer buffer used at 100v for 1hr30. Licor blocking buffer used. SECONDARY ANTIBODY Invitrogen Alexa Fluor 680 goat anti rabbit, 1/5000 in 50:50 PBS: Licor blocking buffer and 0.1% Tween. Incubated for 1hr at room temperature. Washed in PBS-T (0.1%). Final wash in PBS. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No ADDITIONAL NOTES Please could a blocking peptide be supplied as the western is very clean

ANSWER:

Thank you for contacting Abcam regarding ab52492.


I am sorry that you are experiencing some difficulties detecting the correct molecular weight band in WB with this antibody. I have reviewed the protocol information you provided and I am not sure that any changes to this would improve your results. Have you completely denatured and reduced the protein?I would recommend boiling for10 minutes and be sure you have included DTT or beta-mercaptoethanol in the sample buffer.


Another explanationis that this protein is subject to post-translational modifications, acetylation in particular at 10 different residues, resulting in a shift of molecular weight.

Unfortunately a blocking peptide is not available, but I can provide the protein sequence if you were interested in synthesizing your own.


I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions or concerns.

I would use this antibody for rat tissue (IHC-P). However in the Abcam catalog, another antibody is suggested for the same application (i.e.Ab23375). Could you suggest me which would be the best antibody for this application?

Thank you very much

ANSWER:

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

I can confirm that both antibodies recognize rat ALDH1A1, however the immunogens used to raise these antibodies were different. Whilst, for ab23375 a synthetic peptide conjugated to KLH derived from within residues 450 to the C-terminus of - rat - ALDH1A1 was used; for the other antibody ab52492, the immunogen peptide corresponding to residues near the C-terminus of - human - Aldh1a1 was applied.

I hope this helps and if I can assist further, please do not hesitate to contact me.

What dilution and antigen retrieval method should be used for IHC?

ANSWER:

Thank you for your phone call today.

I contacted the lab and was given the protocol used for IHC testing. Antigen retrieval was performed using pH 6 citrate buffer for 20-30 minutes in a heated rice cooker. The exact dilution of this antibody for IHC is variable, but I recommend starting with 1:100 dilution.

I have attached the full protocol to this email.

I hope this information is helpful, but please let me know if you have further questions.