I am new to 2D gel electrophoresis. I tried to follow the basics but the results have come as an absolute shocker. As a newbie my troubleshooting skill is as limited as my hands on skill in the technique.
Protein was extracted from adult rat brain using 7 M Urea, 2 M Thiourea, 4% CHAPS, 10 mM Tris and soluble fraction obtained from supernatant of 100000xg. The protein was precipitated with TCA and pellet solubilized in rehydration buffer. 200ug protein was used during passive rehydration ( overnight at 20 C). IEF was conducted on a GE system followed by equilibration both in DTT and Iodoacetamide(15 min each).
A 12% gel was run for 7 hrs at 100 V and stained using silver staining technique.
Really perplexed where I could have gone drastically wrong. Please help!
There are a number of things I do ignore about your experiment, so I will try to make an educated guess.
Your IF gel seems to have severe defects at pH gradient formation.
If you are using commercial strips, the strip could have been badly rehydrated. If these are home made gels the pH was not allowed to form before the protein load.
As a consequence of the lack of uniformity in hydration, rather steep pH jumps seem to have formed at to point or your strip and your protein may have agglomerated at the defined points were these pH jumps happened.
You need to be very careful, follow all the recommended steps and give them enough time at the rehydration steps, and the desalting IF protocols.
Sometime it is recommended to duplicate the initial steps of voltage ramping in the IF running scheme.
Best wishes,
Rogelio
Hi,
Although I'm probably not able to diagnose what went wrong in your experiment, I am curious about a couple of things:
What pH strip (range) did you use?
What did the strip look like after rehydration? Did you use a blue colorant to check whether the protein-containing solution equally spread over the strip? What did the integrity of the gel at the ends of the strip look like?
Did you place it in the focusing apparatus correctly (cathode/anode-wise)?
Hope this helps and I wish you best of luck in a next attempt! I still lively recall the disappointing feeling of seeing a 2D gel image develop that tells you that the work you have been doing in the preceding days was useless (even though I haven't run a 2D gel for over 10 years)..
First of all, you have good start ;) 2DE is not easy and there can be lots of diffcult points. Good advise was given by Robelio and by Eef. Have you used ready-prep strips? What kind of pH range? Maybe there's plethora of proteis below and above the pI you have used? What about the IEF process? Have you noticed some preciitates around electrodes? If yes, you should change the wet paper wicks often. Have you dried the pellet after TCA precipitation properly? I had similar effects - proteins aggregated at the accidic and the basic end of gel, however, I had also proteins in the middle of the gel. I had pH range 5-8 and someone told me that was because of proteins below and obove the aplied pI.
This could be due to improper IEF process, and also it seems you have used a low pH range strips. Try to use high pH range strip for your sample. probably, this could solve the problem.
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