How to increase protein concentration before loading onto 1D SDS PAGE

Increase protein concentration on PVDF membrane
Alphalyse
Jun 7. 2016

Question:

May I precipitate with TCA before loading to SDS gel?

Is it ok to precipitate my samples with TCA before loading them onto the SDS-Gel – to increase the protein concentration? If not, how do you recommend reducing the volume of my sample?

Answer:

Different methods exist to increase the protein concentration before loading onto a 1D gel, including dialysis, micro con spin filters, precipitation, and chromatography [1, 2].

You can use TCA precipitation, but TCA is a powerful acid and may lead to hydrolysis and fragmentation of some proteins [3].

We often use ice-cold ethanol/acetone precipitation because it increases the protein concentration and removes most salts and buffer components [1-3].

Briefly, the samples are mixed with ice-cold ethanol (100 ul:400 ul) and incubated at -20°C. Subsequently, 400 ul ice-cold acetone is added and incubated at -20°C. We then centrifuge the samples and wash them with acetone/ethanol/water (2:2:1) [2, 4].

Or use this classical “Acetone Precipitation Protocol” to increase protein concentration:

1. Cool the required volume of acetone to -20°C (we keep a bottle in the freezer).

2. Place the protein sample in an acetone-compatible tube. Test the tube before use because some tubes dissolve in acetone!

3. Add four times the sample volume of cold (-20°C) acetone to the tube.

4. Mix the tube and incubate for 60 minutes at -20°C.

5. Centrifuge the tube for 10 minutes at 13,000-15,000 x g.

6. Decant and adequately dispose of the supernatant. Be careful not to dislodge the protein pellet.

Optional: If additional precipitation cycles are necessary to remove the interfering substance, then repeat steps 2-5 before proceeding to step 7.

7. Allow the acetone to evaporate from the uncapped tube at room temperature for 30 minutes. The protein may not dissolve again but stay as a white pellet. Do not over-dry the pellet, and do not speedily vac dry for too long.

8. Finally, you should resuspend in an appropriate buffer.

Most samples can be redissolved in a 1D sample buffer that contains SDS.

Read more about protein separation by Electrophoresis here.

2D PAGE preparation for MALDI ISD

References:

[1]          Posch, A.: “2D PAGE: Sample Preparation and Fractionation,” Methods in Molecular Biology2008

[2]          Chen et al.: A modified protein precipitation procedure for efficient removal of albumin from serum,” Electrophoresis, 2005

[3]          Koontz, L.: “Chapter One – TCA Precipitation,” Methods in Enzymology, 2014

[4]          Zellner et al.: Quantitative validation of different protein precipitation methods in proteome analysis of blood platelets, “Electrophoresisis, 2005

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