Anti-KAP1 antibody - ChIP Grade (ab3831)

Thank you for kind advises about #ab3831.

I have informed your comments to our customer. You recommend following modifications on his protocol.

To decrease the background, try blocking with BSA rather than milk. I also suggest decreasing the concentrations of both the primary and secondary antibodies and decrease the incubation periods. In addition, increase the number of washes.

They have already changed these condition, however these modification did not improve his results.

Additionally, you also suggested running a positive control, and HepG2 whole cell lysate is recommended as positive control. But, please tell us which is a positive control of the KAP1 antibody in mouse extract, because they use a extract from mouse cell line NIH3T3. And please tell us which samples from mouse were used in your test if you possible. I'd like to know whether #ab3831 certainly work on mouse sample.

Please let me know.

Thank you.

ANSWER:

Thank you for your email. This antibody was originally characterized for application in Western blotting using human samples (HepG2 whole cell lysate). We received feedback from a customer who successfully used ab3831 with mouse samples (mouse embryos) in Western blotting and IF. You may read this review by clicking on the reviews tab located on the online datasheet. You may also send an email to the reviewer by clicking on the name.

As I previously mentioned, I would suggest using HepG2 whole cell lysate as a positive control. On the online datasheet for ab3831, there is also a nice Western blot image showing the results with this lysate.

Please let me know if you need further assistance.

We received a compliant for #AB3831 from our customer who bought it in this month. The Lot#s was #87823.

They demonstrated the Western Blotting Assay using this antibody as primary antibody. Our customer reported that signals were not observed at all.

Their experimental conditions was as follows.

Mouse NIH3T3 cell line was used as sample. They prepared the sample as crude whole cell lysate. 10-20ug of protein was included in their sample. Sample ware not induced in their expression.

Membrane was blocked by 5% skim milk for one hour at room temperature.

About primary antibody Dilution ratio was 1:2000. Reaction was performed for over night at room temperature.

About secondary antibody; HRP-alfa-goat IgG, F(ab')2 fragment specific (Jackson) Secondary antibody was diluted up to 10000 fold. They could get good results in other experiment using this secondary antibody.

Background signal was high and they also detect non-specific band, however they could observe only week band of their sample.

Their sample was stained by other primary antibodies ( Anti-BAF155(R-18), goat poly Ab, Santa Cruz; Anti NUP50, goat poly Ab, Abcam ) in same method , however they didn't use positive and negative control.

And they hope that it is replaced with other product (#ab10483) if possible. Could you send another product as replacement?

Please let me know your concern.

Thank you.

ANSWER:

Thank you for your email and I'm sorry to hear that your customer is experiencing difficulty with ab3831. At this point I would like to make some suggestions to try to assist your customer. To decrease the background, try blocking with BSA rather than milk. I also suggest decreasing the concentrations of both the primary and secondary antibodies and decrease the incubation periods. In addition, increase the number of washes. I also strongly suggest running a positive control. Ab3831 was characterized using HepG2 whole cell lysate, which is recommended as positive control.

If these suggestions do not help, please let me know.

I would like to ask for some technical information about your KAP-1 antibody (goat polyclonal, cat no. ab3831). From western blot it seems KAP-1 migrates as a 120kDa band. However its molecular mass should be 88.5kDa. Do you have the reason why it shifts differently from its calculated mass? Thanks.

ANSWER:

Thank you for your enquiry.

According to Swiss Prot and Ugene the predicted molecular weight of this target should be approximately 100 kDa. We have updated the relevant public facing site in order to reflect this data.

However, we would like to emphasize that this antibody has been tested and characterized on HepG2 (human hepatocellular cancer cell line). According to Swiss-Prot database, there are alternative splice variants and isoforms which may cause shift in the band size.

We would like to thank you for bringing this matter to our attention.

I would like to know if this antibody cross reacts with TIF 1 alpha please?

ANSWER:

By BLAST analysis I can find no significant similarity between the epitope used to generate this antibody (Peptide with sequence SSQELSGGPGDGP, from C Terminus of the protein sequence according) and any epitopes in TIF 1 alpha. I therfore think it is highly unlikely that the antibody cross reacts with TIF 1 alpha.