NIH/3T3 nuclear extract lysate (ab14874)

Thank you very much for contacting us with your enquiry.

I'm sorry to hear that there were no bands in Western blot using this antibody. I would expect any of the MEF cell lines to work as a positive control, but I suggest using a whole cell lysate (ab14874 is a nuclear fraction) since that is what we have tested with ab80. What kind of samples are you using other than the MEF lysate?

If you'd like to send some more details about your protocol ( how much protein was loaded, dilution of antibody, blocking solution, etc) I'd be happy to take a look and see if we have any suggestions to improve the results.

I look forward to hearing from you. If there's anything else that we can do for you, please let me know and I'll be happy to help.

Thank you very much for your reply and for the additional information.

Have you tried any other exposure times? Since the specific band seems to be the strongest (with 1:100 of antibody and 15-30 ug of protein), you can probably reduce the exposure time, which will help reducebackground as well. I'd also recommend using a 5% milk block to further reduce non-specific binding. If these suggestions don't improve the results, I'd be happy to send a replacement antibody or issue a credit or refund if you'd like.

Please keep me updated about any new results, and if you have any questions or need anything else let me know and I'll be happy to help.

Have a nice day.

Thank you very much for contacting us with your question and for sending the image of your results.

According to the UniProt page for this protein, there aren't any known post-translational modifications like cleavage or glycosylations that might result in multiple bands-

http://www.uniprot.org/uniprot/O08760

The human protein is known to have several isoforms, but the C-terminal regions are very different and probably wouldn't react with the antibody ab14874. So, these other bands are most likely non-specific. I also ran a BLAST of the immunogen region against the mouse proteome, and no proteins other than OGG1 are predicted to react with the antibody based on these results. Due to this information, we are not sure of the identities of these extra bands, but there may be ways to reduce them.

Would you mind telling me a bit more about the protocol that was used?

1) How much lysate was loaded per lane?
2) What kind of blocking solution was used?
3) How long was the exposure time?
4) How was the lysate stored before being loaded onto the gel?

I look forward to hearing from you. We do fully guarantee the results of this antibody, so I can send a replacement antibody or issue a credit or refund if the results are not satisfactory. Please let me know if you have any questions or if there is anything else that we can do for you.

Thank you for contacting us. We have three antibodies that may be suitable for your purposes:

ab13665: rabbit polyclonal to EZH1 validated in WB on mouse and human samples
ab86128: rabbit polyclonal to EZH1 validated in WB and ICC/IF on mouse and human samples
ab64850: rabbit polyclonal to EZH1 validated in WB, IHC-P, and ELISA on human and mouse samples

Please let me know if you need any additional information or assistance.

Thank you for sending the image of your blots.

When you use the PVDF membrane, are you pre-wetting it with methanol? Because PVDF is hydrophobic, it may not properly bind the blocking agent without the pre-wetting step. This could potentially cause the black blot you are seeing.

The speckled signal observed with the donkey anti-rabbit HRP secondary and 5% donkey serum blocking agent is typically a product of contamination in the blocking buffer or primary antibody diluent. This may be prevented by either filtering your reagents or using fresh buffers. This could also bea result of contamination on the sponge in your transfer apparatus. Thoroughly washing the transfer equipment should prevent this.

This EZH1 antibody is validated using 5% BSA in TBST as a blocking agent, but it should be suitable for use with milk and donkey serum as well. Because the signal observed is so strong, I would recommend using a much higher dilution of secondary antibody (at least 1:50k - 1:100k). Increasing the blocking time to overnight at 4C may also help. If you are looking to try an alternative blocking agent, gelatin is sometimes used. There are also several commercial blocking agents available (including ab126587 and ab64234), however these tend to be casein based. Because casein is the major protein in milk, they may or may not produce different results in your experiment.

I hope this helps, please let me know if you need any additional information or assistance.

Thank you for your phone call. As we discussed, it is possible that your 6 x 15 minute washes are washing the blocking agent off your membrane. It may help to reduce the wash time to 3 x 5 min, shorten the primary antibody incubation to 1 hour at RT, and use less primary antibody. I hope this will help to improve your results, if not, please let me know and I will be happy to help you further.