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Non-Reduced Western Blot

SDS PAGE WESTERN BLOT OXIDATION Non Recuded Electrophoresis

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#1 Adam Erickson

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Posted 01 August 2012 - 08:49 AM

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

#2 DocHazard

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Posted 02 August 2012 - 04:51 PM

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

#3 mdfenko

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Posted 03 August 2012 - 06:51 AM

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.
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#4 Adam Erickson

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Posted 04 August 2012 - 11:08 AM

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!

#5 mdfenko

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Posted 06 August 2012 - 01:46 PM

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.
talent does what it can
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i do what i get paid to do

#6 Adam Erickson

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Posted 07 August 2012 - 10:32 AM

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





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