Anti-Vitronectin antibody [BV1] (ab7165)

Thanks for the response. We are sure about the quantity of vitronectin in the sample because the smaple serum was enriched for the experiment. I used 1:200 dilution and incubated more than 2 hrs. Usually any antibody gives some signal at this dilution. Also I exposed the film for more than 20 min. Again the blot was reprobed with other antibody with similar reagents including sec. antibody, whcih gave positive result. We are sure there no problem with the rest of the reagents except primary antibody.

I would apreciate if you can provide a different lot of ab7165 or another antibody to vitronectin. Expecting your positive response

ANSWER:

We regret to hear about your problems with ab7165 monoclonal antibody to human vitronectin.

The antibody is described in the following article: Zanetti, A et al; Clustering of vitronectin and RGD peptides on microspheres leads to engagement of integrins on the luminal aspect of endothelial cell membrane. Blood 1994, 84: 1116

The researcher of this article use human plasma vitronectin, which was purified under nondenaturing conditions as described in: Preissner, K et al; Physicochemical characterization of human S-protein and its functions in the blood coagulation system. Biochem J 1985, 231: 349.

This may suggest that the concentration of vitronectin in the serum samples is too low to detect. To check this statement we would like to advise you to do a spotblot.

We hope this information will be helpful to you. If you have further questions please do not hesitate to contact us.

Follow up to previous enquiry:

Thanks for the response. We are sure about the quantity of vitronectin in the sample because the smaple serum was enriched for the experiment. I used 1:200 dilution and incubated more than 2 hrs. Usually any antibody gives some signal at this dilution. Also I exposed the film for more than 20 min. Again the blot was reprobed with other antibody with similar reagents including sec. antibody, whcih gave positive result. We are sure there no problem with the rest of the reagents except primary antibody.

ANSWER:

I have contacted the source of the antibody to find out if they can recommend a protocol.

Could you please confirm you used protease inhibitors and kept the samples cold at all times during your experiment prior to boiling your samples with loading buffer?

I have been informed you also have problems with detecting kininogen and I am wondering if you have enough of those two proteins in your purified samples? Do you have a positive control to test whether these antibodies work? I would recommend trying a long incubation (18h) and more concentrated antibody.

I will forward to you any information I receive from the source of the antibody, my apologies for the delay.

Thank you for your patience,

Please let me know how you get on,

BATCH NUMBER 74939 ORDER NUMBER 58763

DESCRIPTION OF THE PROBLEM No signal or weak signal There was no signal in blots (2 seperate blots)

SAMPLE Human serum, purified, 125 ug /blot

PRIMARY ANTIBODY Abcam, ab7165-50, Mouse monoclonal (BV1) against human vitronectin(0.1ug/ul), Diluted in 2% milk in TBST, 1:400 and 1: 200 dilutions, incubated 1-2 hrs, washed 10 min x 3 times in TBST

SECONDARY ANTIBODY Santacruz biotechnology, Goat anti mouse IgG-HRP (0.4 ug/ul), 1:20000, Diluted in 2% milk in TBST, incubated 1 hrs, washed 10 min x 3 times in TBST.

DETECTION METHOD ECL, Super signal pico stable peroxide solution from PIERCE. treated for 2-3 min, Exposed to film for 2 min and and 10 min and there was no sginal on the blot.

POSITIVE AND NEGATIVE CONTROLS USED I have reprobed the same blot with another antibody with similar reagents and I get good results.

ANTIBODY STORAGE CONDITIONS Antibody stored at -20 degree C. Antibody was stored in the storage buffer supplied and diluted in freshly prepared 2% milk solution in TBST prior to incubation.

SAMPLE PREPARATION (Serum purified by Agilent immuno affinity column, (enrcihed by depleting high abundant fractions like albumin, IgG, haptoglobin, transferin etc)

AMOUNT OF PROTEIN LOADED 125 ug / blot

ELECTROPHORESIS/GEL CONDITIONS SDS PAGE, Tris glycine buffer, reducing gel, 8-16%

TRANSFER AND BLOCKING CONDITIONS Wet transfer using Biorad Trans blot apparatus, 4 hrs at 55 V, Tris glycine with 10% methanol, blocked in 5% milk

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? I tried 2 different experiment with 2 blots and 2 dilutions. I do similar methods with several other antibodies from abcam and other vendors and they work OK

ADDITIONAL NOTES I have repeated 2 times with two different blots. I used 0.5 ug/ ml and 1 ug/ml dilution of the primary antibodies. It looks like the primary antibody is not at all binding. I have done parallel experiment with other antibodies and work well. Let me know ASAP what else I can do on this. Thanking you

ANSWER:

I'm sorry you are having problems with ab7165 and would like to thank you for taking the time to give me details of your protocol, it is very useful.

I would like to suggest incubating overnight at 4C and try also more concentrated antibody.

Could it be possible that you have low levels of vitronectin in your samples which cannot be detected by western blot methods? Do you have a positive control to help determine this? Do you use your secondary antibody with other antibodies? If not, could this be the problem?

Degradation of vitronectin could also be the problem. Could it be degraded during the purification process and do you have protease inhibitors in your lysis buffer?

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

Since we are interested in detecting the beta subunit of the vitronectin receptor (alpha v, beta 3), we would like to know: a) which integrin subunit your monoclonal antibody for vitronectin is able to recognize? b) whether the same antibody cross-react with the murine epitope?

ANSWER:

The subunit recognise by this antibody is undetermined, as is it's cross-reactivity with mouse.