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I am using 2D gel electrophoresis to visualize polyubiquitinated proteins. However, while I can see actin and heat shock protein using when appropriate antibodies, I cannot visualize them using anti-ubiquitin antibody. Could it be degradation problem?

I run isoelectric focusing at 200 V overnight (at least 18 hours) and 700 V the last 30 minutes. I am worried that I am applying more volts than needed, because the standard protocol is 200 V for 20 minutes, 450 V for 15 minutes, 750 V for 15 minutes, 2000 V for 30 minutes, but due to problems with the power unit I cannot do this.

I am sure I have polyubiquitination because I use a drug that triggers polyubiquitinated protein accumulation inside the cells.

I would like to add also that cells are collected by scraping in cold PBS and then they are placed in ice and the pellet is also collected at 4 degrees to prevent protein degradation.

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    $\begingroup$ Do you have protease inhibitors in your lysis buffer? If so, which ones? Also, does your anti-ubiquitin antibody work when doing a straight SDS-PAGE Western blot? $\endgroup$
    – MattDMo
    Commented Jul 1, 2015 at 13:26
  • $\begingroup$ MattDMo, My anti-ubiquitin antibody works doing Western blot. $\endgroup$
    – Bio
    Commented Jul 1, 2015 at 13:39
  • $\begingroup$ MattDMo, My anti-ubiquitin antibody works doing Western blot, but I don't use lysis buffer, after collect my cells by scraping using PBS I centrifuge the eppendorf and I freeze the dry pellet (withouth PBS) in liquid nitrogen, then I resuspend the pellet directly in rehydration buffer (urea, CHAPS, ampholytes, bromophenol blue, DTT) and I add Urea and vortex until I see a litle urea precipitates at the bottom, then I centrifugate it at 10.000 rpm 5 min and I use directly the supernadant to rehydrate my strips. $\endgroup$
    – Bio
    Commented Jul 1, 2015 at 13:55
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    $\begingroup$ Do you have a positive control you could use next to your samples? $\endgroup$
    – Chris ♦
    Commented Jul 1, 2015 at 14:00
  • $\begingroup$ That is what I am looking for, I am trying to show that is possible to distingue polyubiquitination from negative control, but I have never done this before, so I don`t have anything to compare as a positive control, if so, that would mean that finally the procedure works and the spots appears. $\endgroup$
    – Bio
    Commented Jul 1, 2015 at 14:45

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There are stable ubiquitinated proteins in mammalian cell lysates even if active proteosomes exist in cells.

First, you might want to make sure that the antibody is applicable to WB.

Then, you would ask if your WB system using the antibody works. You could optimize the condition using just 1D SDS-PAGE followed by WB.

For the condition for isoelectric focusing, when focusing is completed, you get very high electric resistance, so you might want to monitor current after you change the voltage to 700 V. Current decreases gradually, and get stable at some level. But I do not know you can see stabilization by 700 V.

You also might want to check if major proteins such as actin form spots nicely at the expected pI. That could tell if forcusing is completed roughly.

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  • $\begingroup$ Yes, the antibody is applicable to WB because I have used it before. I use a kind of homemade circuit connecting my system to electrodes that are connected to a water recipient, so the current I think is ok. The proteosome is alterated by the drug I use, so the proteins are accumulated in the cells. So if I put more time that the procol say, it could be that a problem because polyubiquitinated protein could be more sensitive to be destroyed or some reason? $\endgroup$
    – Bio
    Commented Jul 2, 2015 at 8:29
  • $\begingroup$ Urea is strong denaturing effects, so you would not have protease activities in the rehydration buffer. I am not sure about proteasome activities during freeze-dry process. Maybe ok, but no guarantee. Can you directly add the rehydration buffer to cell pellets? Or you can add acetone or 10%TCA to precipitate proteins so that proteases are immediately denatured and inactivated, and proteins you are interested in can be protected from degradation. $\endgroup$
    – 243
    Commented Jul 2, 2015 at 18:31

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