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Protein Gel Resolution - Unable to see proteins with a molecular weight less then 14 kDa (Nov/30/2010 )

Im having a difficulty getting good resolution on a protein gel. See the attached image. Im running e. coli whole cell lysate on a 15% resolving gel for 40 minutes at 200 volts run in tris-tricine running buffer. As you can see in the image, i do not see any protein bands which are under 14 kDa. This makes it rather difficult to determine if my protein is being overproduced. Has anyone else encountered this problem? DO you think i should try a 20% resolving gel, or do you think it may be a problem with the buffers i use to make the gel. Any help would be greatly appreciated. If you have a protocol that works particularly well for small proteins let me know.
Attached Image

-bored scientist-

with what are you lysing the e coli? sometimes sample buffer components will disrupt the smaller proteins.

you can test for this by adding some lysis buffer to the standards.

-mdfenko-

Im lysing in 1 x TES (20 mM Tris, 5 mM EDTA, 10 mM NaCl) and lysing by freeze thawing using liquid nitrogen. I do about 6 cycles of freezing and thrawing. Would it be better to use lysozyme?

-bored scientist-

Hello Bored Scientist,

I have a few comments/suggestions that may help in your situation.
1. I donít think that it would be a benefit to move to a 20% resolving gel. If the proteins are not appearing on a 15% resolving gel they wonít appear on the 20%.
2. It is possible that the proteins below 14 kDa are low abundance proteins and do not appear on a Coomassie stained gel. Two quick methods to check this would be to do a dilution series where the first lane is overloaded for the high molecular weight proteins and still stain with Coomassie or try a more sensitive stain, such as silver stain.
3. However, I think that you are not getting good lysing of low molecular weight proteins with your current lysis protocol. There are several commercial lysis kits that get a very high recovery of proteins at all molecular weights. One in particular is ProteaPrep Cell Lysis Kit, Mass Spec Grade from Protea. I predominantly use this cell lysis buffer due to its ability to degrade the surfactant following lysing of the cells. This allows it to then be used with both 2D gel electrophoresis and mass spec.
Hope you find this helpful.

-proteaHeather-

bored scientist on Wed Dec 1 01:50:41 2010 said:


Im having a difficulty getting good resolution on a protein gel. See the attached image. Im running e. coli whole cell lysate on a 15% resolving gel for 40 minutes at 200 volts run in tris-tricine running buffer. As you can see in the image, i do not see any protein bands which are under 14 kDa. This makes it rather difficult to determine if my protein is being overproduced. Has anyone else encountered this problem? DO you think i should try a 20% resolving gel, or do you think it may be a problem with the buffers i use to make the gel. Any help would be greatly appreciated. If you have a protocol that works particularly well for small proteins let me know.



How about running ur gel for (A) Voltage at constant rate while ur mA is 16 per gel?? I usually do that for protein at the size of 12 kD. besides, I boiled my sample for 10 mins and spin it down for 1 min.

-boginei-