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Problem in protein preparation in 2-D electrophoresis - (Apr/23/2006 )

Dear all,

i'm using whole bacterial cell extracts which are prepared by sonication for 2-D electrophoresis. Horizontal streaking are observed. I've tried to use amersham 2-D clean-up kit to remove the impurities and it did improve the result. But still some horizontal streaking are observed. I wonder should I try to modify my protein extraction protocol.

Here is how I extract the protein:
1. the culture broth are centrifuged for 5 min at 4oC.
2. the pellet was washed 4 times with milliQ water and resuspend in rehydration buffer (7M urea, 2M Thiourea, 4% CHAPS).
3. The solution was then sonicated 7 times on ice (60s with 90s break, 50% max. output).
4. Then centrifuged at 10000g for 20min at 4oC (to remove cell debris, RNA and DNA?)
5. The supernatant was then stored at -80oC and are loaded as protein sample.
The protein sample was cleaned using 2-D clean-up kit before each round of 2-D

I've seen some protocol had used DNase and RNase to remove RNA and DNA, I wonder if it is really an important step as some protocol said RNA and DNA can be removed during step 4 of my protocol. Or are there any important steps i've missed? Please advise~

THanks a lot^^

-shicaeon-

hello,

as long as you're sonicating, the DNA/RNA shouldn't be an issue. still, there are a few things you can try....

if you're using a 2D cleanup kit, go ahead and spin your cleaned-up sample at 100,000 x g for about an hour to make sure you're not getting streaking due to un-dissolved particulate. also, what proteins are you looking for? are they soluble, or membrane proteins (or are you just not sure at this stage). if you're only looking for soluble proteins, i'd suggest extracting in a "soluble protein buffer," clean that up and run it- you'll get less streaking than if you include membrane proteins. if, on the other hand, you want membrane proteins, you would perform the extraction as above (to remove the cytosolic proteins...hey, why keep them around if you don't need them?), but keep the pellet (which contains your membranes/membrane proteins) and re-extract in your IEF buffer. this can be subject to a 2D cleanup and run out on your IEF strip.

if you really are after total protein, just focus on ultrafuging your sample and see if that doesn't minimize your problem. also, i think biorad or amersham sells a product called "de-streak," or something like that.

i hope this helps.



QUOTE (shicaeon @ Apr 23 2006, 08:05 AM)
Dear all,

i'm using whole bacterial cell extracts which are prepared by sonication for 2-D electrophoresis. Horizontal streaking are observed. I've tried to use amersham 2-D clean-up kit to remove the impurities and it did improve the result. But still some horizontal streaking are observed. I wonder should I try to modify my protein extraction protocol.

Here is how I extract the protein:
1. the culture broth are centrifuged for 5 min at 4oC.
2. the pellet was washed 4 times with milliQ water and resuspend in rehydration buffer (7M urea, 2M Thiourea, 4% CHAPS).
3. The solution was then sonicated 7 times on ice (60s with 90s break, 50% max. output).
4. Then centrifuged at 10000g for 20min at 4oC (to remove cell debris, RNA and DNA?)
5. The supernatant was then stored at -80oC and are loaded as protein sample.
The protein sample was cleaned using 2-D clean-up kit before each round of 2-D

I've seen some protocol had used DNase and RNase to remove RNA and DNA, I wonder if it is really an important step as some protocol said RNA and DNA can be removed during step 4 of my protocol. Or are there any important steps i've missed? Please advise~

THanks a lot^^

-johanski-