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Resuspension of Protein after TCA Precipitation

tca precepitation resusprension protein

Best Answer labtastic, 14 August 2014 - 08:01 AM

I have not encountered this problem myself, but if I did, these would be the things I'd think about:

 

1. not over drying the TCA/acetone-washed pellet before adding sds sample buffer (I take my acetone-washed pellets and dry them at 95C for exactly 1 min [no more] with the eppendorf tube open)

2. once the sds-sample buffer is added to your pellet, vortex for a minute then boil at 95C for 5 min. 

3. add in reducing agent to your sds-sample buffer if you haven't already

4. consider adding in 8M urea to you sds-sample buffer

5. Are you actually trying to dissolve your pellet in 4x sds sample buffer? You should be resuspending it in 1x sds sample buffer...

6. I was a little unsure in your post...is it your purified protein that you are having issues resuspending, or the cell extract? If it's the cell extract, then I wouldn't be too concerned as long as you've got your protein sufficiently separated from it and you can run that on a gel properly.

7. Also, you don't have any potassium salts in your buffer do you? potassium-sds is insoluble. Though TCA ppt'ing the protein in the K salt should remove the that problem.

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#1 ags2014

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Posted 11 August 2014 - 04:47 PM

So, I have a recombinant his tagged protein being expressed in e coli. So, we grow a culture of E coli, spin it down and separate the cells from the media/supernatant. The cells are lysed and the lysate run over a nickel column. Purification is not an issue, but the analysis of the proteins is.

 

When I mix the lysate or the Nickel column flow through with 4xSDS loading buffer (commercial) we end up getting some kind of precipitate. We initially thought, DNA crashing out. So we thought, a clean up is necessary.

 

We performed TCA precipitation (followed by washes with acetone). We tried resuspending the pellet in 4xSDS loading buffer, and we have the same issue - the protein will simply not dissolve. So what we thought earlier to be DNA crashing out, is actually protein that doesn't seem to like 4xSDS.

 

So, we added some elution buffer (nickel resin elution - tris, imidazole and salt based at pH 8.0) to the mix to see if we could dissolve it - but it remains as a precipitate. 

 

Have you observed anything similar where the ppt refuses to dissolve in the 4xSDS after TCA precipitation? If so, how did you get around it?

 

 



#2 labtastic

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Posted 14 August 2014 - 08:01 AM   Best Answer

I have not encountered this problem myself, but if I did, these would be the things I'd think about:

 

1. not over drying the TCA/acetone-washed pellet before adding sds sample buffer (I take my acetone-washed pellets and dry them at 95C for exactly 1 min [no more] with the eppendorf tube open)

2. once the sds-sample buffer is added to your pellet, vortex for a minute then boil at 95C for 5 min. 

3. add in reducing agent to your sds-sample buffer if you haven't already

4. consider adding in 8M urea to you sds-sample buffer

5. Are you actually trying to dissolve your pellet in 4x sds sample buffer? You should be resuspending it in 1x sds sample buffer...

6. I was a little unsure in your post...is it your purified protein that you are having issues resuspending, or the cell extract? If it's the cell extract, then I wouldn't be too concerned as long as you've got your protein sufficiently separated from it and you can run that on a gel properly.

7. Also, you don't have any potassium salts in your buffer do you? potassium-sds is insoluble. Though TCA ppt'ing the protein in the K salt should remove the that problem.







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