Anti-Furin antibody (ab3467)

So, you are saying, the 60kD band is the Signal Sequence (˜24AA)+ Propeptide (˜130AA) + catalytic domain (˜280)+ partial RGD domain.



Thank you,

ANSWER:

Sorry for this misunderstanding.

We believe that the antibody recognizes the mature form of the protein. The pro-peptide is expected at 87kDa and once the signal and pro-peptide are cleaved off the protein, it migrates at 63 kDa.

I hope this is helpful and wish you a good weekend.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 111548

DESCRIPTION OF THE PROBLEM Antibody recognises multiple bands - major ones at 250, 100, 75 (triplet) and also picks up 37 kDa MW marker! Peptide (ab4989) takes out 250, upper band of 75 kDa triplet and MW marker (!).

SAMPLE Human cultured cardiac fibroblasts - whole cell homogenate

PRIMARY ANTIBODY Abcam furin convertase (ab3467) 1:500 - 1:1000

DETECTION METHOD Pierce ECL

POSITIVE AND NEGATIVE CONTROLS USED HT-1080 cell homogenate

ANTIBODY STORAGE CONDITIONS -20C aliquoted

SAMPLE PREPARATION Laemmli sample buffer + PMSF + Na vanadate. Boiled + 2-mercaptoethanol for 5 min.

AMOUNT OF PROTEIN LOADED 50-100 ug per lane

ELECTROPHORESIS/GEL CONDITIONS 10% gel. reducing.

TRANSFER AND BLOCKING CONDITIONS Blocked in 5% Milk in TBS Tween

SECONDARY ANTIBODY [a competitor] rabbit-HRP 1:2000

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Antibody dilution, addition of neutralising peptide

ADDITIONAL NOTES Attached is a figure of blot with and without peptide.

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. We have received a favourable review of our Furin Convertase antibody (ab3467) albeit with acknowledgement of the detection of some non-specific bands.

We often receive complaints relating to aberrant bands that can often be resolved with the use of 5% BSA as a blocking agent. We find it gives a cleaner more specific blot. I would therefore like to recommend its use on this occasion.

I would also recommend performing an overnight incubation of the antibody, at a more dilute concentration e.g. 1:1500 at 4oC as again we often find that this increases the specificity of the blot.

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