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Protein Purification from Qiagen RNeasy Kit


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#1 thebigpiro

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Posted 05 January 2010 - 08:40 AM

I am wondering if anyone has done this and has any tips or tricks. Essentially, it's an acetone precipitation from RLT buffer lystate. Here's the short protocol:

http://www1.qiagen.c...ls/pdf/RY22.pdf

I find this to be a convenient way to obtain protein from cells and tissues for use in SDS-PAGE, and it's economical from a tissue standpoint. But I have two main problems with it:

1) The pellet is very hard to resuspend and I generally have to use a rotor-stator homogenizer to get the job done, even using an SDS resuspension buffer.

2) My protein yields seem low compared to my RNA yields.

Is there any way to make this work better? Alternatively, Qiagen sells an AllPrep kit for DNA/RNA/Protein, and while I don't need the DNA, I would check the kit out if protein yields are better and if it's easier to work with.

#2 jah

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Posted 05 January 2010 - 09:28 AM

I've tried the acetone-precipitation method a few times...if you can set up a parallel experiment and extract the protein and RNA separately I'd advise against it. That said, I did learn a few things in the course of it all....

1) The pellet is very hard to resuspend and I generally have to use a rotor-stator homogenizer to get the job done, even using an SDS resuspension buffer.


Two things can help with the 'insoluble pellet'. I do an extra wash in 70% Ethanol to remove the residual salts which are difficult to solubilize and can cause problems with the electrophoresis. You can also try sonicating the pellet in the SDS-PAGE buffer. Another way is to resuspend the pellet in a small volume of 2D page buffer (e.g. 0.1% CHAPS/7MUrea/2M Thiourea/10mM DTT/40mM Tris pH8.5), and let it stand at RT for 30mins before adding your SDS loading buffer. Do not heat proteins in a Urea-based buffer, due to carbamylation of lysines.

Is there any way to make this work better? Alternatively, Qiagen sells an AllPrep kit for DNA/RNA/Protein, and while I don't need the DNA, I would check the kit out if protein yields are better and if it's easier to work with.

I tried the AllPrep and was not very fond of it. My main problem was that it was that the lysis buffer precipitated on ice, and that the desalting column (sephadex?) was not easy to work with. The RNA was fine, but the protein was sparse, and was more work than just setting up a parallel experiment collecting the protein into a more appropriate buffer.

#3 thebigpiro

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Posted 05 January 2010 - 10:11 AM

Thanks, jah. To clarify, were your protein preps worse with AllPrep than with RNeasy followed by acetone precipitation or were they similarly bad with both?

I have not done the ethanol wash step in the past, but I'll try it and see if it helps. I've considered sonication as well but have used a homogenizer since we have one handy in our lab. Our typical protocol also calls for a page sample buffer using Tris HCl, glycerol and SDS but I may try your urea solution as well.

#4 jah

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Posted 05 January 2010 - 05:54 PM

The preps weren't worse per se both worked just fine for a western, just more work than they needed to be, when I could just 'lyse & load' Good luck




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