Evaporation of TEAB buffer - Problems with viscosity/residue

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AnaC
Proton Member
Proton Member
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Joined: Mon Sep 17, 2012 8:39 pm

Evaporation of TEAB buffer - Problems with viscosity/residue

Postby AnaC » Tue Sep 18, 2012 9:52 pm

Hi there,
I've been trying to figure out a sample prep protocol for an iTRAQ experiment for a while now, and have recently run into a problem when using speedvac to concentrate my protein samples.

I'll try and keep this simple - I'm interested in the soluble/intracellular proteins in my sample, so we developed a protocol using a hypotonic shock/lysis to extract these proteins of interest. My buffer is composed of 1mM DTT, 5mM TEAB and 1X Complete protease inhibitor (EDTA free). After the lysis and I've spun out the insoluble protein component, I top up the sample with a more concentrated buffer to get a final sample buffer composition of 1mM DTT, 500mM TEAB and 1X Complete.
At this point I have about 100-130ug of soluble protein in 1.2mL of buffer, which I need to reduce to <50ul for the iTRAQ labelling reaction. Then I concentrate the samples by speedvac and do the downstream labelling, digestion, peptide fractionation etc. I had tested parts of this method and worked it all the way through to ms/ms once. We chose to use 500mM TEAB buffer as this is the buffer used throughout the iTRAQ protocol.

However, I've been trying to repeat my experiment to get more data and I'm having issues at the early point where I concentrate my sample from ~1.2mL to 40ul. It seems to proceed smoothly until the sample is concentrated to about 100ul, but at that point the sample becomes quite viscous and aerated (like bubbly spit, sorry for the gross analogy!) and then very soon after this point it dries completely leaving a reasonable quantity of bubbly off-white residue. It will readily dissolve in water, but it extremely viscous - which means something is going wrong...
At first I thought this may be excess protein, but I have triple checked and my protein assays seem to be correct and I don't believe I have enough protein to cause this issue. The samples are sonicated also, so DNA shouldn't be causing the viscosity.

Does anyone have any ideas as to what might be causing my issues? My mass spec technician/research fellow didn't think I should proceed with the iTRAQ labelling as things are, I didn't want to waste $ on the labels as I can easily produce more samples.

Any thoughts at all would be welcome! Cheers, Ana.

Infinity
E. Coli Lysate Member
E. Coli Lysate Member
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Joined: Wed Dec 21, 2011 8:22 pm

Postby Infinity » Wed Sep 19, 2012 5:13 am

Well, evaporating you samples in the presence of huge concentartion of TEAB and DTT (which are not volatile) is not a good idea. Why don't you digest your proteins first, then desalt peptides and only then concentrate on speedvac? This seems more logical especially considering that you have to label with iTRAQ your peptides (not proteins).

Infinity
E. Coli Lysate Member
E. Coli Lysate Member
Posts: 107
Joined: Wed Dec 21, 2011 8:22 pm

Postby Infinity » Wed Sep 19, 2012 2:15 pm

Oups, TEAB should be volatile - sorry for this (I mixed it with TEAP). If you want to label proteins you could try to precipitate them with acetone or TCA.

Biomarker
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Joined: Thu Aug 09, 2012 3:28 am

Postby Biomarker » Thu Sep 20, 2012 1:42 am

Hi,

Go for buffer exchange into 0.5 M Triethyl ammonium bicarboante (TEAB) using spin concentrators with 5 kDa or 3 kDa MW cutoff (Agilent Technologies or Sartorius (Viva Spin 4 ml; using the same)). Do it with three rounds (Usually two rounds are enough) of buffer addition, with centrifugation at 4000 rpm at 4 C for 24 mins every time. At the end concentrate till you will get 40 ul with some additional pulses of 3 or 6 mins.
This is working fine for me....

Cheers.........
Biomarker
:)

Biomarker
Albumin Member
Posts: 88
Joined: Thu Aug 09, 2012 3:28 am

Postby Biomarker » Thu Sep 20, 2012 5:42 am

Hi,

You need not have to even go for the buffer exchange. Just concentrate the sample in Viva Spin 4 ml till it reduced to 40 ul.

Biomarker


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