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

N- and C-terminal sequencing from PVDF membrane
Alphalyse
Jun 7. 2016

Question:

I have another question:  Is it ok if I precipitate my samples with TCA prior loading to the SDS-Gel? If not, how do you recommend  to reduce the volume of my sample?  Thanks

Answer:

There are different methods to increase the protein concentration before loading onto a 1D gel, including: dialysis, microcon type spin filters, precipitation, and chromatography.

You can use TCA precipitation, but TCA is a very strong acid and may lead to hydrolysis and fragmentation of some proteins.

We often use ice cold ethanol/acetone precipitation, because it increases the protein concentration and removes most salts and buffer components.

Briefly, the samples are mixed with ice cold ethanol (100 ul:400 ul), incubated at -20°C. Subsequently, 400 ul ice cold acetone is added and again incubated at -20°C. The samples are then centrifuged and washed with acetone/ethanol/water (2:2:1).

 

Alternatively, you can use this classical “Acetone Precipitation Protocol”:

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

2. Place the protein sample in 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 properly dispose of the supernatant. Be careful to not dislodge the protein pellet.

Optional: If additional cycles of precipitation are necessary to completely 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. Do not over-dry the pellet and do not speedy vac dry for too long. The protein may not dissolve again, but stay as a white pellet.

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

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

 

Read more about 1D and 2D protein separation here.

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