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How to extract protein from SDS-PAGE gel?


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#1 zzll

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Posted 10 July 2009 - 05:45 PM

Hi all,

Does anyone know how a protein band can be extracted from SDS-PAGE gel w/o using any commercial kit?
The gel is stained by coomassie bright blue.

Many thanks.

zzll

#2 Nrelo

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Posted 10 July 2009 - 09:26 PM

Hi all,

Does anyone know how a protein band can be extracted from SDS-PAGE gel w/o using any commercial kit?
The gel is stained by coomassie bright blue.

Many thanks.

zzll


Are you going to do mass spectrometry? There are procotols on the internet for dissolving the stained gel. You can cut your target band out and dissolve it.

#3 T C

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Posted 11 July 2009 - 01:05 AM

Hey,

Just cut the band and put it in a dialysis tubing with an appropriate cut off, seal the two ends and pass voltage, the protein will move out of the gel but will stay in the tubing due to the cut off. Take the buffer out of the dialysis bag and concentrate to get yr protein.

Hope it helps

Best,
TC

Hi all,

Does anyone know how a protein band can be extracted from SDS-PAGE gel w/o using any commercial kit?
The gel is stained by coomassie bright blue.

Many thanks.

zzll



#4 zzll

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Posted 11 July 2009 - 06:56 AM

Thanks a lot, Nrelo and TC.


To Nrelo,

Yes, I want to analyze the protein using Mass spectroscopy. What kind of buffer should I use? I googled yesterday but didn't find useful info. Maybe I didn't use the correct keywords.

To TC,

Could you please tell me how I can apply voltage on the dialysis tubing? Same question, what buffer shall I use? Or it doesn't matter?

I used precast minigel. Is the protein on the gel enough? Shall I use multiple bands or do a big gel?

I'm so happy that I get valuable answers here. Many thanks!

zzll

#5 T C

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Posted 12 July 2009 - 01:59 AM

Okay......... I thought u want it for immunization.....for mass spec analysis you do an in gel trypsinization, its done in ammonium bicarbonate. There are standard protocols. Just search the net for "in gel trypsinization" and you would find it. Applying voltage on a dialysis bag is a crude setup in which you perform electrophoresis like a DNA gel, just that yr gel is replaced by the dialysis tubing which is totally submerged in the buffer.

Best,
TC

#6 zzll

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Posted 12 July 2009 - 04:13 AM

Thanks a lot, TC.

I guess I probably misled you by saying mass spect. Actually, I still need the intact protein. I'm doing expression of a protein from insect cells. I found an interesting band so I want to know its molecular weight before I continue with large scale expression.

So I won't need the in-gel digestion. But I know how to apply the voltage on the tubing from your post. Can I just use typical TAE buffer for this purpose?

Thanks again!

zzll

Okay......... I thought u want it for immunization.....for mass spec analysis you do an in gel trypsinization, its done in ammonium bicarbonate. There are standard protocols. Just search the net for "in gel trypsinization" and you would find it. Applying voltage on a dialysis bag is a crude setup in which you perform electrophoresis like a DNA gel, just that yr gel is replaced by the dialysis tubing which is totally submerged in the buffer.

Best,
TC



#7 T C

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Posted 12 July 2009 - 04:57 AM

Hey,

Don't you already know the approximate molecular weight since u run it on the gel? If you plan to run the intact protein on the mass spec, it has to be a small protein that can run in the linear mode or else you need to perform in gel trypsin digestion.

The buffer should be the same buffer that is inside the dialysis bag and the buffer that you want yr protein to be in.

Best,
TC

Thanks a lot, TC.

I guess I probably misled you by saying mass spect. Actually, I still need the intact protein. I'm doing expression of a protein from insect cells. I found an interesting band so I want to know its molecular weight before I continue with large scale expression.

So I won't need the in-gel digestion. But I know how to apply the voltage on the tubing from your post. Can I just use typical TAE buffer for this purpose?

Thanks again!

zzll



#8 zzll

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Posted 12 July 2009 - 06:19 AM

The problem is the protein of interest isn't at exactly the same position as the reference lane. I don't know if this is due to slightly different migration rate or protein itself. So I want to have a MALDI. I know this protein will perform well using MALDI.

Now I have an idea what I should do next.

Thanks a lot, TC

zzll



Hey,

Don't you already know the approximate molecular weight since u run it on the gel? If you plan to run the intact protein on the mass spec, it has to be a small protein that can run in the linear mode or else you need to perform in gel trypsin digestion.

The buffer should be the same buffer that is inside the dialysis bag and the buffer that you want yr protein to be in.

Best,
TC

Thanks a lot, TC.

I guess I probably misled you by saying mass spect. Actually, I still need the intact protein. I'm doing expression of a protein from insect cells. I found an interesting band so I want to know its molecular weight before I continue with large scale expression.

So I won't need the in-gel digestion. But I know how to apply the voltage on the tubing from your post. Can I just use typical TAE buffer for this purpose?

Thanks again!

zzll






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