I am targeting staph aureus with my molecule to see protein profile in treated and untreated staph
i am here with attach my recent 2D gel picture please suggest me how to improve it
protein extraction is done by sonication and acetone precipitation.
Is there any other way to see the difference in protein profile before and after treatment by molecule?
I think your first dimension is not separating properly, probably do to some chemical contaminant (salt, Tris or similar). Please provide more information - did you wash the cells after harvesting (and if so in what)? What did re-suspend in for sonication? etc...
After harvesting culture in LB broth i did not wash the cell (Missed the step)
I emulsified in Lysozyme solution prepared in distilled water
How ever after sonication i have added acetone in supernatant (chilled) centrifused and collected the pellet and emulsified in re hydration buffer
Do you have suggestion for my protein extraction from gram positive staph aureus?
A few ideas that will hopefully help:
1. Wash cell pellet twice with ice-cold buffer TE (10 mM Tris, bring to pH 8.0 with HCl
1 mM EDTA) and twice with ice-cold DI water, to remove salt and buffer.
2. Why Lysozyme ? isn't sonication enough? I would suggest collecting the cells in small volume, say 5 ml original to 100ul DI water, and sonicate 5 times 20 seconds + 40 minutes delay. Make sure sonication is on ice. BTW is the Lysozyme in buffer?
3. Centrifuge - 4C, 5 min., 8000g to remove unbroken cells and cell debris.
3. Precipitate in 80% acetone (add 4 volumes and keep at -20C for ON). Centrifuge 20 min, ~14,000g.
4. Resuspend pellet in Thiourea/urea/CHAPs loading buffer and run your 2D.
The problem, you will miss some cell-wall proteins etc. You might try skipping the the sonication and acetone precipitation and simply dry the resuspend cells (remove all liquid, centrifuge again for 1 min, remove more liquid, repeat, then leave at dry incubator for a 10 minutes). Then resuspend semi-dry pellet in loading buffer.
Good luck.
Yoram
@ James John,
Can you please mention the name of 2D apparatus? If its Ettan IPGphor, then I would suggest you few moves.
Ya, I do it regularly. By looking into the gel, it seems that the 1st dimension run was doubtful either with your sample or with the run time. Was your dye successfully run across the anode? If the dye didn't move completely, then you need to do two things:
1. Increase the time for IEF, or
2. It is due to high concentration of buffer salt in your sample, which need to be reduced.
Ah, Sorry, that is not possible as I have recently finished by lab responsibilities and winding up my thesis for PhD degree. Apart from this, you have different instrument (BioRad) in which you need to ask the technical expert from the company. I can only assist you in troubleshooting.
Sorry. The most likely problem is salt and/or Tris in your sample, so wash your samples throughly before lysis! Also, you should probably add iodcetamide to your loading buffer. Finally, use only high quality reagents for your 2D, the IEF part is very sensitive.
@Yoram Gerchman
I agree with you sir. If there would be high salt concentration, then it won't be possible to do complete IEF. If he had moved into the 2nd dimension (SDS-PAGE), then we can assume that his strips would have focussed properly.
Precipitate with 80% Acetone. Wash your cells in DI 3 times, break them using sonication on ice, [Optional but recommended : centrifuge slow speed to remove unbroken cells and cell debris], add 4 volumes of acetone and keep at -20C overnight, centrifuge maximum speed for 20 min, 4C, remove liquid and dry pellet (MOST proteins will be in the pellet). Resuspend in 2D loading buffer.
Yeah, acetone precipitation will take care of your high salt concentration as only proteins will precipitate out. Resuspend the pellet in loading buffer or Lammeli buffer. This will take care of your protein.
OK
Did you pre-wash the cells in deionized water?
What did you break the cells in and how?
Centrifuge to remove unbroken cells and cell derbies?
Did acetone percepitiaion and re-suspended in what buffer?
How much kV/h did you accumulate? What did the program look like?
Yoram
Have you checked the dye font in your Immobiline pH gradient strip after the IEF? The dye font should be disappeared in the strip after complete run.
IEF is the crucial step. Sometimes, the sample could not finish its run in the time scheduled in the program. Hence, the time is increased to finish the 1st dimension separation.
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