Anti-PACS1 antibody (ab56072)

OK I will run the gel again to see if increasing the boiling time helps. If I reply to this email in a week or so, will I be able to get in touch with you or will this thread be closed?

ANSWER:

If you reply to this email, it will come straight to me, the thread will not be closed. So please get back to me whenever you get your results and I will be happy to help further if I can.
Good luck in your experiments.

So I loaded the same amount of protein in all lanes as you can tell from the b actin loading control. The reason that some of the bands are fainter is because I was evaluating the efficiency of PCAS1 siRNA in HeLa cells, so those are different siRNAs. I think the reason why the blot is confusing is because the bands above 150 are quite prominent compared to the ones above 100. So you think the ones above 100 are the real bands?

ANSWER:

Thank you for your reply.
Since the band closer to 150kDa is decreasing in intensity as you use siRNAs against PCAS1, then I would say that is the correct band. You could try boiling your samples for longer, up to 10 minutes, as the protein may be in a complex and boiling longer may help.
Please let me know if there is anything else I can help you with.

Yes I boil them in loading buffer which also has DTT for 6 min at around 95C. Sorry I forgot to mention that.

ANSWER:

Thank you for your reply.
I just have one further question, is there a difference in how much protein were loaded into each well of the gel, as in the lane closest to the marker, the band is intense. That signal then decreases as the lanes get further from the marker, but the band at just above 100kDa stays relatively constant.
I look forward to your reply.

I recently bought the Anti-PCAS1 Ab (ab 56072). I used this Ab to detect PACS1 expression in HeLa cell lysates. As you will see from the attached file, I see multiple bands and so I am not really sure which one is the correct band. According to the product insert, the band size is supposed to be around 105 kda, but the band I see at around 150 kda is stronger than the rest, which is confusing. Briefly this is the protocol I followed:
1. Lysed HeLa cells in EBCD buffer which has DTT.
2. Loaded samples on a 7% denaturing SDS PAGE gel
3. Used semi-dry transfer at 23 V for 1 hr and 20'
4. Blocked with 5% BSA in TBST for an hr at RT
5. Probed with 1 µg/ml Ab in 5% NFDM in TBST overnight at 4C.
6. Washed with TBST for an hour at RT (multiple washes)
7. Probed with 1:2500 Goat Anti-Rabbit HRP conjugated secondary Ab for an hr at RT
8. Washed for an hour with TBST (multiple washes)
9. Developed the blot with a 1:1 dilution of Femto (Pierce) for 30s.

ANSWER:

Thank you for contacting Abcam.
I just have a question about the protocol that you are using; do you boil your samples before loading them onto your gel? I ask as maybe the protein is complexed in some form and so that is why you are seeing a slightly higher molecular weight. Also this protein does have a number of phosphorylation sites, which will cause a small increase in the molecular weight.
I look forward to your reply.