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Lack of bands in SDS-PAGE gel - (Jul/16/2007 )

Hi, I am currently running SDS-PAGE gels to determine expression of a GST-tagged viral protein (48kDa). Basically, I induce BL21 cells with IPTG after growth reaches O.D. 0.6, harvest cells after 4 hrs by centrifuging at 10,000 rpm for 2 minutes, and carry out lysis by vortexing with 8M urea for 5 min. Then, I centrifuge at 10,00rpm for 10 min to collect the supernatant, which is added to the gel (12%).

Previously, I followed this protocol, and I observed my protein of interest quite clearly. This week when I ran the gels, the lanes are very faint after staining, and its almost impossible to discern my protein band. I am really baffled by this, and would be really grateful if somebody could help me figure out why this is happening.

My supervisor says the cell lysis was not proper, since all lanes are missing/ very unclear, but I did a Bradford assay prior to loading into the wells, and I calculated I was loading around 40 ug of total protein - is it possible that the assay is giving wrong values (cell debris or something messing it up?)
The only other change I did this week was during cell lysis, I vortexed for around 25 min (rather than the said 5 minutes). Would this cause protein degradation or precipitation into the pellet?

The only other reason I can think of is that the staining solution (Connassi e stain) is not working properly - especially since its being reused in our lab. Has anyone else had previous problems with this?

I would be really thankful for any suggestions or solutions...
Regards
Akshay

Edit: i just remembered, could it be possible that the 8M urea has 'expired'? I realize that we are using it from a 500ml bottle, need it be freshly prepared?

-Neo7-

first of all tell me whether after lysis u centrifuged at 1000 rpm or 10,000 rpm.

-rachana bhatt-

sorry, i centrifuged at 10,000 rpm after lysis to get the supernatant

-Neo7-

missing, uncler or smeary bands speak for (partial) proteolysis in your preparation;

CBB solution can be re-used several times; more sensitive is silver-staining

-The Bearer-

QUOTE (Neo7 @ Jul 16 2007, 08:08 AM)
Hi, I am currently running SDS-PAGE gels to determine expression of a GST-tagged viral protein (48kDa). Basically, I induce BL21 cells with IPTG after growth reaches O.D. 0.6, harvest cells after 4 hrs by centrifuging at 10,000 rpm for 2 minutes, and carry out lysis by vortexing with 8M urea for 5 min. Then, I centrifuge at 10,00rpm for 10 min to collect the supernatant, which is added to the gel (12%).

Previously, I followed this protocol, and I observed my protein of interest quite clearly. This week when I ran the gels, the lanes are very faint after staining, and its almost impossible to discern my protein band. I am really baffled by this, and would be really grateful if somebody could help me figure out why this is happening.

My supervisor says the cell lysis was not proper, since all lanes are missing/ very unclear, but I did a Bradford assay prior to loading into the wells, and I calculated I was loading around 40 ug of total protein - is it possible that the assay is giving wrong values (cell debris or something messing it up?)
The only other change I did this week was during cell lysis, I vortexed for around 25 min (rather than the said 5 minutes). Would this cause protein degradation or precipitation into the pellet?

The only other reason I can think of is that the staining solution (Connassi e stain) is not working properly - especially since its being reused in our lab. Has anyone else had previous problems with this?

I would be really thankful for any suggestions or solutions...
Regards
Akshay

Edit: i just remembered, could it be possible that the 8M urea has 'expired'? I realize that we are using it from a 500ml bottle, need it be freshly prepared?


hi,
i think your cell lysis is not proper. after dissolving in the lysis buffer u should add 200ug/ml lysozyme for 30 mins and then sonicate the for 5 min. after that you collect supernatant by centrifugation at 10000 rpm. alternatively, if you dont need to check solubility of your protein , you can take the cell plate in water and boil it for 5to 10 min then collect supernatant after centrifugation at 10000 rpm. hope this will work
good luck

-veerubiotech-