At the moment, I'm struggling with a very basic technique. I want to run proteins from lysed T-REx HEK293 cells on a SDS gel. However, instead of sharp protein bands I just see a smear after staining it with instant blue.
I'm using precast gels (4-20%) and I also tried different buffers. At the moment, I'm using RIPA buffer (Tris buffer with NaCl) + 1% Triton X + cOmplete Mini Protease inhibitor tablet. My protocol for lysing the cells is the following: I remove the media, place them on ice, wash with ice-cold PBS and scratch the cells from the flask with lysis buffer. Subsequently, I perform three freezing-thawing cycles on dry ice and in a 37C water bath. In the end, I spin the cell debris down at 14,000rpm for 10min and use the supernatant for my gel. I boil a fraction of the proteins with SDS lysis buffer before I load it on the gel.
I haven't faced this problems before, although I was working with these cells for quite a while already.
Many thanks for your ideas!
Hi, there are two things to try, add 10mM or even higher DTT in your lysate before you boil them for gel. Second thing if your protein is highly concentrated you may see smear, try various concentration of sample in parallel on the gel, the one with lower concentration will help give sharper bands. These two methods will clear the smear and get a sharp band on the gel. Best wishes
Thamarai
Hi
In addition to the Thamarai comments, you must run the gel slowly in ice chamber.
This may be resolve your problem.
Since I have not performed a protein analysis of this kind in over 20 years, I'd ask a few questions:
1. Are you trying to isolate all of the cellular proteins on an SDS gel? If so, use a very small quantity of total protein to the well and use a more sensitive staining technique. Keep the gel running at 4ºC.
2. Are you trying to isolate cytosol proteins and not the interamembranous-bound poteins? If so, consider performing another step in differential centrifugation to remove lysed membranes and mitochondria (40 K at 30 minutes to an hour---been a while since I did this protocol but there should be a protocol you can find. It's one I did for my thesis 35 years ago).
3. One of our greatest contaminants in performing SDS PAGE of total cellular proteins from certain WBCs was that there was a huge amount of DNA contamination that caused smearing. You might consider that as a possibility. Additional differential centrifugation or a longer centrifugation period may let you eliminate most of that as well.
Keep in mind though, I've been teaching most of the last 20 years and not running a lab! ;-)
In addition to Prof. Marks comments No. 3. Try to remove to the DNA contaminantion by the addition of Dnase and incubate @ 37 C for 30 min. So that your problem will be fixed.
If you want to know its because of DNA contamination. Reaction mixture will become turbid and sticky to your tubes. You may also see the changes after the addition of Dnase.
All the best..
With regards to Prof. Ramachandran's statement: we used DNAse successfully as well back in the 1980's, but make sure you account for that in your protein analysis after the electrophoresis. I wasn't sure if there was any newer method to remove DNA or the enzyme from a sample.
Hi Simon,
I completely agree with Thamarai, Masood, Mark and Shiyam. Two additional simple recommendations would be to:
1) Use fresh running buffer. The more you re-use running buffer, the less resolution you will see on your gel after staining.
2) Try decreasing/removing salt (NaCl) and detergent (Triton) from your samples before loading. These two have an effect on protein gel migration.
All the best!
High voltage can also cause smearing. try to decrease the voltage. Contamination of DNA is also one factor though.
Thank you all for your help! I solved the problem by changing several different bits of my protocol. If the details may be helpful for you, please let me know and I'll forward them. :)
Hi Simon Satter, I am interested in the solution, I am suffering the same problem not with the cell lysate but with the supernatant itself, my r-protein is a secretory one.
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