Anti-MiTF antibody [C5] - ChIP Grade (ab12039)

I have 2 questions concerning Mitf ab 12039 C5 clone:
- What is the specificity of his antibody, does it recognise only Mitf
isoform M or also Mitf Isoforms A, H, and C?
- In the datasheet is written that the immunogen is N-terminal fragment
of human microphthalmia protein. Is this the N-terminal fragment of
different isoforms of Mitf or of only 1 isoform? And if of only 1
isoform, which one was used?

ANSWER:

Thank you for your patience.

The following information was obtained:

The Mitf antibody ab12039 [C5] recognizes both human and mouse Mitf in melanocytes. It is a mouse IgG1 Kappa chain antibody. It isbelieved to beM form specific, but wouldneed to be validated that as the literature was to utilize the antibodies C5 and D5 for diagnostic purposes in melanomas. The antibody was originally made from an N terminal peptide coupled to KLH.

I hope this information is of some help to you. Please do not hesitate to contact us if you need any more advice or information.

I have 2 questions concerning Mitf ab 12039 C5 clone:
- What is the specificity of his antibody, does it recognise only Mitf
isoform M or also Mitf Isoforms A, H, and C?
- In the datasheet is written that the immunogen is N-terminal fragment
of human microphthalmia protein. Is this the N-terminal fragment of
different isoforms of Mitf or of only 1 isoform? And if of only 1
isoform, which one was used?

ANSWER:

Thank you for contacting us.

I have been in touch with the lab. We need to check with the originator of the antibody for these details. I will let you know as soon asI hear back.

Thank you for your patience.

I performed all the changes you suggested and am still unable to get a band for MITF using the (C5)mouse monoclonal antibody using an even wider panel of melanoma lines and primary human melanocytes. Once again, I am able to get a band for survivin and beta-actin using the same lysates. Could you send me a replacement antibody for MITF from a different lot of the C5 monoclonal?

ANSWER:

Thank you for your e-mail and informing me you have tried the suggested modifications to your protocol, I appreciate the amount of work you have performed and have immediately arranged a replacement vial to be sent to you.

The order is reference 172077 and will be shipped to you today,

Could you please let me know how you get on with the replacement vial?

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 166189

DESCRIPTION OF THE PROBLEM Non-specific bands obtained. Specific bands between 52-70 kD not seen.

SAMPLE 3 primary melanocyte lines, and 3 melanoma lines lysed in 2% SDS lysis buffer.

PRIMARY ANTIBODY MITF (C5)mouse monoclonal antibody by abcam at 1:1000 dilution in 5%milk for 90 minutes, followed by 3 washes (30 minutes) in PBST. As control, beta actin mouse monoclonal (sigma) and survivin rabbit polyclonal (Dr. Altieri) used similarly.

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED The survivin and beta-actin blots as noted above

ANTIBODY STORAGE CONDITIONS antibody aliquoted and stored at -20C. Thawed on ice, used at 1:1000 dilution for western.

SAMPLE PREPARATION protease and phosphatase inhibitors (sigma) used. Tris used to pH at 7.4. sample heated at 94C for 3 minutes.

AMOUNT OF PROTEIN LOADED 30 micrograms

ELECTROPHORESIS/GEL CONDITIONS 10% SDS-PAGE

TRANSFER AND BLOCKING CONDITIONS 1X Tris-glycine, 20%methanol used as transfer buffer. Transfer carried out on ice 100V for 1 hour.

SECONDARY ANTIBODY [another company] goat anti-mouse (and anti-rabbit for survivin) used at 1:10000 in 5%milk. incubated for 30 minutes, followed by 3 washes in PBST for 30 minutes

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? tried lengthening the incubation and wash steps

ANSWER:

I'm very sorry to hear you are experiencing problems with ab12039 and thank you for providing your protocol details so we can look into your problem.

I would like to recommend the following modifications to your protocol:

-use a RIPA or NP40 lysis buffer (the recipe can be found, if you need it, on our protocols page on our website) so as to extract optimally the protein, which is in the nucleus (leaving the samples to lyse for 2-4hours in the lysis buffer containing plenty of fresh protease inhibitors)

-block the membrane in 5%BSA in TBST (Tween 20 0.1%) for 1 hour, then rinse lightly the membrane 10 seconds in TBST and incubate the antibody in TBST only.

-try diluting the antibody more, you may find that with the above changes you can dilute more.

If you still experience problems with the above changes please do not hesitate to contact me and I can arrange for a replacement vial or refund as you purchased the antibody in the last 90 days,

I am sorry to bother you again, but still I did not manage to get any signal from my anti-MiTF C5 (#ab12039) blot, even after new recommended conditions (5% BSA for blocking and 1:250 primary Ab, and additional control: Skmel cell lysate). Did you receive any other enquiries about troubles with this antibody, maybe the same batch (151283)? I wonder if maybe there is something wrong with my vial, or this batch. Would it be possible to get sample from the batch You tested and it is working for sure?

I am pretty desperate to get the right antibodies, as this blot with MiTF is the last one for my thesis and a paper.

Best regards,

ANSWER:

Thank you for getting back to me.

I appreciate the steps that you have taken to try and get this antibody to work.

I have checked our records and we do not have any other complaints with regards this batch. I would be happy to provide you with a replacement vial of a different batch provided that the antibody was purchased within the past 90 days (Lot number: 177646). If this is the case please provide me with your order details including the date of purchase and original order number.

I look forward to hearing from you.