horizontal streaking

[tina]

Horizontal streaking is BIG problem on my 2D gels. I have tried many different variants of our protocol, but horizontal streaking is still present. I am working with protein extract from E. coli.
I have used extraction and rehydration buffer with 10 mM and 65 mM DTT, but there are no significant differences beetwen the gels - horizontal streaking was still present. I was also very careful when working with DTT - I have added DTT to both buffers just before I had used the buffer. So, the possibilty for DTT oxidation was minimal.
Even after the addition of DeStreak Solution (Amersham) instead of rehydration buffer to protein sample horizontal streaking was still present.
I also have used 2d clean up kit which did not affect the horizontal streaking.
Does anybody know why is horizontal streaking still present or has simillar problems?
Thanks

[gandalfthegrey]

Hello Tina,

I usually centrifuge my Ecoli samples at very high speed for a long time to remove insoluble particles. Are you doing this as well?

What are you using to lyse your cells? Sonication?

[danfive]

Check out the 2D troubleshooting guide under horizontal streaking ---> http://www.proteinsandproteomics.org...s_1/pro03.html
Also a lot of times you have to simplify your protein solution this can be reducing protein concentration, using Molecular Weight Cutoffs, Zoom IPG strips (shortened range like pI 4-7, 5-8 instead of 3-10), or prefractionation like Microrotofor isoelectric focusing.
If you want to try different reducing agent, I recommend ProteoPrep Reduction & Alkylation kit (Sigma) it uses TBP instead of DTT.

[dnamolecule]

I found quite a few more as well thanks Danfive.

A few more causes and solutions for Horizontal 2-D gel streaking:

your sample was not completely solubilized prior to 2-D gel loading
solution: Make sure that your sample is completely and stably solubilized

your sample was poorly soluble in the rehydration solution

solution: Increase the concentration of the solubilizing components in the rehydration buffer and/or Increase the concentration of IPG buffer.

your sample has Interfering Substances or Impurities.

solution: Modify or Purify sample preparation to limit contamination of these interfering substances


your sample has High Salt or Ionic Impurities

solution: you can reduce the salt concentration below 10mM by dilution or dialysis of your samples.

Also some more:

1. If you used SDS which is an ionic detergent, it must not exceed 0.25%
2. You can also try loading less sample.
3. Prolong the focusing time and maybe reduce it also.

Please tell us what works
may help us all in the future!

[danfive]

I don't use SDS until the electrophoresis step, in the TGS buffer.
This buffer recipe was passed on to me from a great tech guy at Pierce:
CHAPS lysis buffer:
20mM Tris pH 8.0
2mM EDTA
0.5%CHAPS-added fresh

Incubate on ice 40min with occasional flicking.
Centrifuge at max speed to clear cell debris, transfer supernatant to new tube.

Very simple, very few steps.

[tina]

Originally Posted by gandalfthegrey

Hello Tina,

I usually centrifuge my Ecoli samples at very high speed for a long time to remove insoluble particles. Are you doing this as well?

What are you using to lyse your cells? Sonication?

Thanks.
After sonication (4 X 15 s) in extraction buffer I centrifuge the samples 20 min at 13000 rpm. At which speed do you centrifuge?

[tina]

Thanks everybody. I have no horizontal streaking on my gels anymore since I have used 10 mM Tris pH 7,4 + 250 mM sucrose for washing the biomass.