What are the components or what lysis buffers can be used to extract proteins from cells so as to perform 2D gel electrophoresis?
Dear murlidharan,
For protein extraction, i use 0.1 M Tris buffer containing 7 M Urea, 2 M Thiourea, 2% Triton X-100, 1% β-mercaptoethanol.
What is the sample? Then you can start thinking about extraction solutions.
Thanks nitesh !!! @Matthew padula: i will be using a cell line treated with some compounds as the sample for protein extraction.
Then Nitesh's suggestion will work fine. I would substitute the Triton for 4% CHAPS or 1%C7BzO and then reduce and alkylate with tributylphosphine (5mM or 20mM DTT) and then acrylamide monomers (20mM) for 90 mins. Then make sure you buffer exchange the proteins into 7M urea, 2M thiourea and surfactant (0.5-1% C7BzO or 4% CHAPS) to remove the tris and other contaminants that will interfere with focusing. Precipitating with 5 volumes of acetone works well, then resuspend pellet in 7M urea, 2M thiourea and surfactant (0.5-1% C7BzO or 4% CHAPS).
@ Matthew Padula: Thank you very much for the suggestion. I have read people often use this composition for protein extraction: 9.5 M urea, 2% CHAPS, 0.8%pharmalyte, and 1% DTT. Just want a suggestion regarding this also. And why pharmalyte in extraction/lysis buffer? can we use anything instead of pharmalyte?
thanks in advance !!!
For IEF, 7M urea, 2M thiourea seems to be the most chaotropic (there's papers by Ben Herbert about this). Pharmalyte is the trade name for GE's ampholytes and Bio-Rad also sell them. Adding ampholytes can improve the solubility of some proteins and it doesn't hurt to add it. My personal opinion is to have the protein in the minimum number of additives possible. Urea, thiourea and surfactant are the minimum and I would only add ampholytes after that. Reducing agents are unnecessary if the sample has been reduced and alkylated in solution prior to focusing. They move in the electric field and can interfere with focusing. Make sure you sonicate with an ultrasonic probe to shear up DNA as well.
Best thing to do is to try a set of conditions with a small number of cells and perform a protein assay. Then load ~150ug onto a 7cm pH3-10 strip, focus for 50kVh and run second dimension using a 12% self cast gel. This is the cheapest way to test conditions and you can tweak the extraction chemicals and conditions. Once you are happy, you can move to larger 11cm or 18cm gels for differential display.
@Mathew padula: thanks for that interesting suggestion !!! so that means i can use a combination of 7M urea,2M thiourea, 0.1M Tris,4% CHAPS,1% DTT and protease inhibitors for the preparation of cell lysate. After that an overnight acetone precipitation with 5 times the volume of buffer and redissolving the pellet obtained in the rehydration buffer will enable good focusing of the proteins in the IEF cell. For the clarification regarding the IEF parameters that I am using, I have been using a 3 step protocol :
1: 250 V for 15 minutes
2: 4000V for 30 minutes
3 4000V for 8000V-Hrs
4: 500V hold
Thanks in advance !!!
In addition of the earlier respone, you can use lysis and rehydration buffer :
Composition of lysis buffer : Tris 20mM ( 0 .06057g), Urea 7M (10.5105), Thiourea 2M (3.806g), CHAPS 4%(1g), EDTA 2mM (0.0186g), PMSF 1mM, Triton x100 2%(500µl), Protease Inhibitor cocktail 1µl, DTT 10mM(Should Use After Protein Estimation)for 25 ml
Rehydration buffer: Urea 7M (16.816g), Thiourea 2M (6.0896g), CHAPS 1.2% (0.48g), ASB14 0.4% (0.16g), Ampholyte 1%, Bromophenol blue 0.002% (80 µl) for 40 ml.(DTT and IPG buffer are added just to prior to use 7 mg DTT per 2.5ml RB, Store at -20oC)
Focusing Conditions for GE system, for Biorad you can start with 250 V
Step 1. 500 V for 7 Hour (Slow)
Step 2. 1000V for 1 Hour (Step)
Step 3. 8000V for 3 Hour (Gradient)
Step 4. 10000V for 1:30 Hour (Gradient)
Step 5. 10000V for 1 Hour (Step)
Step 6. 500 Hold.
It worked in my case and for your reference, I attached a figure of 2D-Gel by this protocol where I used 100 ug protein and silver staining
@narendra sharma: thanks a lot for that and i think this will help me a lot !!!!
Narendra's suggestions are spot on. Only thing I do different is to leave out the reducing agent for the focusing. But his gel looks great. What size IPG/gel is that?
@Narendra Sharma: I am wondering whether the six steps mentioned above are used for what kind of IPG strip (i mean of what length and pH)? We are using Biorad IEF cell and up until now we have followed the protocols that is mentioned in the Biorad cataloge. The 3 steps that I have ,mentioned above is used for 7cm pH 3-10 strip. @ Matthew Padula: yes the gel looks great !!!
The voltage profile would work on a 7cm strip. Someone told me once that the higher the numbers of volts per cm, the "tighter" the proteins will focus. I always focus at 10,000V for 11cm IPGs. Normally:
1) 150-3000V over 3-5 hours (concave ramp)
2) 3000-10000V over 5-8 hours (linear ramp)
3) 10000V until 100kVh is reached (But for a 7cm this could be 50kVh)
Hello Dr. Narendra Kumar Sharma, it will be of immense help if you can share the detailed protocol of 2D Gel electrophoresis that you follow for your work with mammalian cell lines. Actually I am a newbie and the first person to do 2D gel electrophoresis in my lab. Could you please help?
Thank you.
Hello, can i ask some question?
I performed 2D gel in 7cm strip follow this condition
step 1. 300 V for 200 VH
step 2. 1000 V for 300 VH(Gradient)
step 3. 5000 V for 4500 VH (Gradient)
step 4. 5000 V for 3000 VH
but i have some problem with instrument, it run only step 1 and 2.
i will ask that how should i do? can I use this strip run IEF again or i should to perform new experiment?
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