Anti-Bax antibody (ab7977)

i have questions due to ab7977 anti Bax. i stain murine liver to check apoptose with ab7977, but i get many times stained nuclei in liver , it should to be stained cytoplazm. could you give me please support and information .

ANSWER:

Thank you for contacting us. I am sorry that ab7977 is producing incorrect staining on your mouse liver sections. Could you please provide some additional details of your protocol?

1) How were your samples fixed and embedded?

2) Did you perform any permeabilization or antigen retrieval? If so, what buffers and conditions were used?

3) What blocking agent was used, at what concentration, and using what incubation time?

4) What were the primary and secondary antibody dilutions and incubation times? What secondary antibody was used?

5) Did you run a no-primary antibody control?

6) It would be very helpful if you could please provide an image of your staining. What is your original order or PO number?

I will be happy to assist you further once I have this additional information.

Dear Madam/Sir,    I am Shereen El Mashad from Cork cancer Research Center , Ireland  and I have inquiry regards BAX antibody  I want to detect the active form but I don’t know between the 2 antibodies BAX6a7 and BAX(n-20) which one you recommend to detect the active form?    THANKS FOR YOUR HELP AND SUPPORT.

ANSWER:

Thank you for contacting Abcam. I have looked at the available information for both antibodies, ab5714 and ab7977. Based on what I have read, I believe that ab5714 would recognize the active form. I am basing this recommendation off the following reference: Hsu YT & Youle RJ Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations. J Biol Chem 273:10777-83 (1998). The above reference showed that the ab5714 antibody does not recognize the soluble form of BAX. If there is anything else I can help you with, please let me know

I am interested in purchasing a selection of antibodies for the study of truncated variants of Bax, Caspase 5 and TGF beta receptor II. The following antibodies may be of use to me, as your datasheets indicate that the N-terminus has been used as the immunogen. However, I need to know which specific residues have been used in order to know whether the antibodies are going to be helpful. I wish to use the antibodies in Western Blot & IHC experiments on colon cancer cell lines.

Bax ab15181 (ab33020, ab5714 & ab7977 all have specific immunogen peptide sequences supplied)

Caspase 5 ab25899 ab40887

TGF beta receptor II ab28382 ab28383

I would also be interested in antibodies to the N-terminus of TAF1B, TCF7L2, IGF2R (ab26938 should be okay) and MSH3. Ideally I would like to order soon to have the antibodies for use by late January.

Thankyou for you help with this

Regards

ANSWER:

Thank you for your enquiry. I have contacted the originators of these antibodies and here is the sequence information I obtained.

ab25899: This immunogen sequence is proprietary, but we can run any alignments with the sequence that you require.

ab40887: The immunogen is 19aa long peptide that lies within the first 50 aas on N-termincal of human Caspase-5.

ab28383/ab28382:LDTLVGKGRFAEVYKAKLKQN (these are the same antibody, one is prediluted and one is concentrated)

ab15181: GSGEQPRGGGPTSSC

Before buying any of these antibodies, I recommend that you check to make sure they are tested in Western blot and IHC and also make sure they have been tested in the sepcies of the sample you are using. We can only guarantee the antibodies if you use them as they are described on the data sheets.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

Thank you for your information about Caspase 3 antibody. I will let you know later. I have rabbit polyclonal anti bax antibody cat# 7977-1 lot# 158757. I have tried 3 times already. There was no significant band. The condition as follows 5% milk blocking, wash 3 times in pbs-t 1% milk, 1:100 dilution primary (bax), wash 3 times, then 1:1000 secondary antibody (HRP conjugated).

It was exposed for 30 min still no band or very faint. Do you have any suggestion

ANSWER:

I'm sorry to hear you are having a problem with ab7977.

It would be helpful if you could give me more details by filling out the questionnaire at the following link:

http://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=7977&mode=questionaire

In the meantime I would suggest the following: 1) Please use HeLa lysate as a positive control as we did on our datasheet, 2) block for 30 min in 5% milk, 3) wash with PBS-T without milk, 4) incubate in primary and secondary antibodies without milk. Please fill out the form so I can see if I have additional advice for you.

Please let me know if this helps and do not hesitate to contact us for further advice.

DESCRIPTION OF THE PROBLEM I have been given this antibody by a medical researcher to perform my routine IHC technique on FFPE tissue sections. His test tissue is sheep kidney. Your product data sheet states that IHC-P is a tested application but there is no recommecnded dilution or antigen unmasking given. Can you give me any idea of a statrting dilution and is unmasking necessary? Thank you.

SAMPLE Rabbit kidney

FIXATION OF SAMPLE Formalin 10%

ANTIGEN RETRIEVAL Do I need to?

ADDITIONAL NOTES Not able to attemp technique until more info is obtained from you. Thnaks.

ANSWER:

Thank you for your enquiry. When testing ab7977 and ab7974 the tissue sections were (briefly) pressure cooked for antigen retrieval in citrate buffer pH 6, for 1 minute. We recommend a starting dilution of 1:50-1:100 dilution. Other antigen retrieval steps may work but the one described above has been used for these antibodies.

I hope this information helps, please do not hesitate to contact us again if you need further advice,

Best of luck with your immunohistochemistry!