Anti-FZR1 antibody [DH01 (DCS-266)] (ab3242)

You asked about a positive control: I transfected a myc-cdh1 plasmid for a positive control in HeLa cells and detected a really strong band both with an anti-myc antibody and with the cdh1 Ab. Both bands were just a little higher than the 62 KD band of the NEB marker I use. However, the lanes of the non-transfected HeLa cells were clean. What I'm struggling with now is why other papers published use the same clone (but the Novus Biologicals antibody) and are able to detect endogenous cdh1 -they are even able to IP it- in non-transfected HeLa cells.

Amount of protein loaded: Yes, you are right I meant to say that I did the IP with 1 mg of protein lysate. The whole cell lysate samples I run are loaded with either 50 or 100 micrograms of protein. Those samples did not show any endogenous cdh1 expression either.

Lysis buffer composition: 0.5% NP-40, 25 mM Tris, 100 mM NaCl, 1 mM Sodium Orthovanadate in addition to a Protease Inhibitor Cocktail from Sigma containing 104mM AEBSF, 10 microM Aprotinin, 2 mM Leupeptin, 4mM Bestatin, 1.5 mM Pepstatin A, and 1.4 mM E-64. The lysis buffer is also supplemented with 20mM MG132 to prevent proteasomal degradation.

If you have any suggestions I would really love to hear about them. I was about to simply purchase the Novus Biologicals antibody cited in the literature but I thought to give your antibody a last try. I hope we can make it work.

ANSWER:

Thank you for your reply and the further details of your protocol. It is very clear to me that the product you received should work and must have been damaged during shipping or storage. If you can please provide your order number I can offer you a replacement vial if the antibody was purchased in the last 90days.

Please note that the Novus antibody is actually from Abcam (Novus used to distribute our products) so if you did purchase from Novus you would still be buying our antibody.

I look forward to hearing from you,

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM Target protein is not precipitated. No bands are observed only the heavy chain of the primary ab.

SAMPLE HeLa cell lysates

PRIMARY ANTIBODY mouse Cdh1, the antibody in question. Cross reacts with human. Added to lysates with NP-40 detergent so that the final concentration present was 2 micrograms/ml. Incubated overnight. No washes were done until after protein G beads were added.

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Positive control was whole cell lysate before immunoprecipitation (but no bands were observed there either). Negative control was an immunoprecipitation with IgG for primary. A band was detected (presumably the Heavy Chain) at the same molecular weight as the ones seen on the test IP samples.

ANTIBODY STORAGE CONDITIONS Stored at 4 degrees celsius

SAMPLE PREPARATION Used lysis buffer with NP-40 detergent. Protease inhibitor cocktail from Roche added.

MATRIX USED Protein G agarose

DETERGENT NP-40

AMOUNT OF PROTEIN LOADED 1 mg

ELECTROPHORESIS/GEL CONDITIONS Reducing, 10% SDS PAGE

SECONDARY ANTIBODY No secondary antibody was added, used protein G agarose beads

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Concentration of Ab (have used 5 micrograms/ml)

ADDITIONAL NOTES A while ago I used the [another company] antibody and it detected cdh1 at a MW higher than the 62 kd marker I use. Now I only see a band above the 47 kd marker. A band of that size is also present in the IgG control IP. That leads me to believe I'm seeing heavy chain (together with the fact that I don't see a band on the Whole Cell Lysates lanes)

ANSWER:

I'm very sorry to hear you are experiencing problems with ab3242.

In order to understand your problem better and to assist you I would be very grateful if you could please clarify a few steps in your protocol:

1) you mention "A while ago I used the Novus antibody and it detected cdh1 at a MW higher than the 62 kd marker I use. Was this the same clone? I only able to find two antibodies on the Novus website, clones DH01 (DCS-266) and AR38.2 (both Abcam antibodies).

2) It would seem that your reprobing/western blotting doesn't work with ab3242 either. Have you tried a positive control in western blotting? We know that Jurkat or LS174T cell lysate will express significantly high levels of cdh1 and therefore should give you a signal.

3) you mention that the amount loaded is 1mg. Do you mean that you IP 1mg of lysate?

4) Can you clarify please what is present in your lysis buffer, how long you lyse the cells for, how long you incubate the lysate with the protein G beads? This is really important information for me to understand your protocol better.

I would agree with you that it appears you are detecting the heavy chain around 50kDa rather than the protein of interrest.

Thank you in advance for providing the above information so I can understand your problem better and resolve this matter, I look forward to hearing from you,

To Whom It May Concern:

I had three questions regarding your cdh1 antibody.

1. Has anyone published a Western using your cdh1 antibody? 2. Which region of the cdh1 protein was the antibody raised? 3. Does this antibody recognize both the phosphorylated and non-phosphorylated versions of this protein?

Thank you so much

ANSWER:

The answers to your questions are as follows:

1) Not to the best of our knowledge. 2) Unfortunately, the exact epitope recognised by this antibody has not been mapped. 3) Probably, but we can't be certain until tested. If phosphorylation occurs at the site of the epitope, we would expect to see at least reduced (if not inhibited) binding.

If the antibody is found not to perform as stated on the datasheet, Abcam will offer a refund or replacement.