Non-Reduced Western Blot - (Aug/01/2012 )

Hello,

Recently I have been trying to detect the oxidized form of a protein. In order to do so we've been trying to use a non-reducing loading buffer. The only difference is the non reducing buffer lacks 2-Mercaptoethanol (also β-mercaptoethanol, BME, 2BME, 2-ME or β-met). 2ME cleaves the disulfide bonds so I know our proteins many not be as denatured, but we've been experiencing problems where our gel doesn't really run at all. We think the proteins may be getting stuck at the top, as if they are grouping together or forming dimers/trimers.

Is there any difference in the non-Reducing protocol and the reducing protocl for anyonee else? Ours are pretty similar. Someone suggested spinning down the samples before loading, but in order to remove dimers/trimers we would need a high powered centrifuge.

Any suggestions on how to get non-reduced western blots to work? Is there any reason that the proteins would be getting stuck, and is there anything we can do about it? Or even is there a better method for detecting the oxidation of a protein?

P.S. We do still boil our samples and everything for the non-reduced.

Thanks,

Struggling Intern

Are you making your own gel or using a purchased precast gel?

for how long do you boil? excessive boiling can cause aggregation of proteins. if you must boil, then you can try 60-70C for 10-20 minutes.

we don't boil when we use non-reducing buffer.

Yes, we are making our own gels. I've heard suggestions about changing the percent ingredients to vary size of pores and makeup of gel, but we're not so sure about that since reducing run just fine.

We boil 5 minutes at 100C and pop the top halfway to prevent gaseous buildup from popping the top (of the eppendorf tubes).

Would boiling longer at that low temperature still server to denature the proteins without aggregation?

Thanks!

if boiling at 100C, you should not pop the tops. that will allow vapor to escape, reducing the volume of the sample in an uncontrolled manner. you should lock the tops to prevent them from popping (you can buy lock-top tubes or cap locks).

longer at lower temperature should denature without aggregation.

Thank you very much! I am about to try that with a gel right now.