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SDS PAGE gel doesn’t resolve my proteins – HELP!!!

SDS PAGE gel doesn’t resolve my proteins – HELP!!! - Molecular Biology Techniques

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Old 09-03-2012, 07:24 PM
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Default SDS PAGE gel doesn’t resolve my proteins – HELP!!!



Our lab is having some troubles withSDS-PAGE gel. We prepare our gels exactly the same way we always did, however, for the past 6 months (yes…six months)…we have experienced a weird phenomena in our running. Our samples, when applied to SDS-PAGE, migrate at the same velocity through the gel and do not separate in different molecular weight. It’s like our gel had not a mesh of acrylamide/bis-acrilamide to separate the different proteins weight. All protein run together during the time course of electrophoreses, even the ladder.
What possible component of electrophoresis may account for this problem? Reagents? Could it be any electrical element?
I attached a picture from my last try onrunning a gel with purified protein from different molecular weight. This gel was stained with Comassie blue. As you can see, all different size of proteins migrates together at the buffer front. Moreover, it seems that all protein tend to diffuse laterally within the gel because we can observe a square weakly stained with comassie.
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Old 09-04-2012, 04:01 AM
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Default Re: SDS PAGE gel doesn’t resolve my proteins – HELP!!!

have you check the pH of your resolving and stacking buffer? Are you still using the same buffer for the pass 6 month? Have you make new stock to try?

Is the gel took longer to polimerize? Or the gel feel less firm than previously? What is the percent of gel did you prepare? You could prepare fresh ammonium persulfate. unless your ammonium persulfate powder is "kinda" wet.

Can you please list out what reagent change and you still experiencing this problem.

Thanks!
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Old 09-05-2012, 07:34 PM
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Default Re: SDS PAGE gel doesn’t resolve my proteins – HELP!!!

Quote:
Originally Posted by butters View Post
have you check the pH of your resolving and stacking buffer? Are you still using the same buffer for the pass 6 month? Have you make new stock to try?

Is the gel took longer to polimerize? Or the gel feel less firm than previously? What is the percent of gel did you prepare? You could prepare fresh ammonium persulfate. unless your ammonium persulfate powder is "kinda" wet.

Can you please list out what reagent change and you still experiencing this problem.

Thanks!

Agree.

Check the pH of the buffers for making the gel as well as the running buffer. Make sure that the stacking and resolving buffers haven't been switched around. Making the resolving gel with stacking gel buffer could create this problem because the proteins would not "de-stack".

4x Stacking buffer (upper gel) should be:
0.5M Tris, pH 6.8
0.4% SDS

4x Resolving Buffer (lower gel) should be:
1.5 M Tris, pH 8.8
0.4% SDS

This looks like a major problem so I don't think it's some thing that is a little off, it must be something seriously messed up.

If you make your own running buffer (tris-glycine-sds) remember NEVER EVER pH this with HCl. The buffer does not need to be pH-ed, if the correct amount of TRis and Glycine are added, the pH will be ok. This will make your gel run very strangely (usually you notice this because the voltage/current is way off from what it normally is).
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Old 09-07-2012, 06:02 PM
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Default Re: SDS PAGE gel doesn’t resolve my proteins – HELP!!!

Hi everyone,

Thanks for replying me.

Regarding your concernings:

1) Yes, we have already check the pH of our buffers (resolvind and stacking), and make them again with our powder and with others lab powder.
2) Our gel take about 2 minutes to polymerize and use it 1 hour after that.
3) We alway make new ammonium persulfate.

4) We use that same recipe you sent for our stacking and resolving buffers.
5) But, we always pH with HCl our running buffer. Could this fact account for our strange running?

I will try to make a new running buffer, without adding HCl...and I´ll reply for you later...
Thanks a lot
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Old 09-08-2012, 05:20 PM
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Default Re: SDS PAGE gel doesn’t resolve my proteins – HELP!!!

Bad news...just run my SDS PAGE with running buffer not adjusting pH and have the same result. I can´t resolve my protein...high and low molecular weight proteins run together trought the gel...they don´t separate...

Any clue?

Really need some help!

Thanks
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Old 09-09-2012, 03:22 PM
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Default Re: SDS PAGE gel doesn’t resolve my proteins – HELP!!!

Quote:
Originally Posted by Snsantos View Post
Hi everyone,

Thanks for replying me.

Regarding your concernings:

1) Yes, we have already check the pH of our buffers (resolvind and stacking), and make them again with our powder and with others lab powder.
2) Our gel take about 2 minutes to polymerize and use it 1 hour after that.
3) We alway make new ammonium persulfate.

4) We use that same recipe you sent for our stacking and resolving buffers.
5) But, we always pH with HCl our running buffer. Could this fact account for our strange running?

I will try to make a new running buffer, without adding HCl...and I´ll reply for you later...
Thanks a lot


Yes adding HCl to the running buffer will definitely mess up the running conditions. But it sounds like you checked this and it didn't help.

How about the sample buffer??? What recipe are you using for your loading buffer.

The other option would be to buy the reagents (gels, buffers etc) from a company. This would be expensive but it sounds like you're spending a lot of time trying to get this to work so maybe it would be worth it
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Old 09-10-2012, 06:04 AM
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Default Re: SDS PAGE gel doesn’t resolve my proteins – HELP!!!

Snsantos,

Did I see 2 minutes for gel to polymerize? What is the percent and vol of APS you are using (w/v)?

Please give list out your buffer preparation formula. Did you prepare the acryl/bis-acryl or was it come pre-prepare?
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