6 replies to this topic

[alex2815]

Hi!
I was tring to make blots for MMP-9 in pig kidney.
The results showed two very faint bands around the appropriate position which were about 84 kda and 92 kda. I also had two strong bands at 25kda and ~60kda. Does anyone explain what are those bands? I used standard protocol from http://www.cellsigna...estern_BSA.html. Any idea to remove those 25 and 60 kda bands?


Thanks!!!

[mdfenko]

the 60 kDa band may be an artifact caused by aged reducing agent (it has been identified as keratins).

[alex2815]

mdfenko, on 16 March 2012 - 11:47 AM, said:

the 60 kDa band may be an artifact caused by aged reducing agent (it has been identified as keratins).

Thank you for you reply!

It is possible. But I have been wearing gloves all the time during the experiments, and these ~60kda bands did not show up on the other blots that I did before, using same reducing agents, buffers and secondary Ab , but with different primary Ab.

The primary Ab I used can recognize the epitope between 107-117 AA in the human MMP-9 sequences. I used that epitope to run a protein Blast with human/pig Keratins, and no similar sequences were found. If those bands are keratins, what things can detect them in my blot? primary Ab or secondary Ab?

Thank you very much!

[mdfenko]

the keratins can be from dust. the primary antibody may not be as specific as it should be or the secondary may be detecting them.

the bands become more prominent as the reducing agent ages.

[alex2815]

mdfenko, on 20 March 2012 - 01:07 PM, said:

the keratins can be from dust. the primary antibody may not be as specific as it should be or the secondary may be detecting them.

the bands become more prominent as the reducing agent ages.

Thank you!

I don't think it is the secondary ab since we have been using the same reducing agent and secondary Ab and the 60kDa bands never showed up. It is probably due to the primary Ab or the protein has been digested. But I have found several papers mentioned that the MMP-9 purified from U937 cells would produce 67kDa MMP-9 via APMA-mediated activation. It said that it is the final form of the MMP-9. Are these papers helpful to explain the situation I have or just useless because MMP-9 activation might be tissue-specific, and they were doing cell studies and I am doing animal studies?

What are those 25kDa bands? Are they also from the reducing agent or just non-specific binding, digested proteins? My labmate and I also get similar non-specific 25kDa bands on some of the other blots.

Thank you!!!

[mdfenko]

the papers appear to explain the situation nicely. both bands (60 and 25 kDa) appear to be proteolytic products of the mmp-9 precursor.

[alex2815]

mdfenko, on 21 March 2012 - 07:43 AM, said:

the papers appear to explain the situation nicely. both bands (60 and 25 kDa) appear to be proteolytic products of the mmp-9 precursor.

One concern is that we cannot be sure about if the MMP-9 acts like this way in vivo.

Thank you very much!