Hello there;
I'm trying to optimize SDS-PAGE/ Western blot condition for Eukaryotic cell membrane. After tissue separation, resuspending in RIPA buffer and sonication, I centrifuge samples at 12000g to sediment nucleus and mithochondria, followed by centrifugation of supernatant at 37000g for 25min to separate membrane. Membrane pellet is then resuspended in PBS+PMSF. 75 micrograms of membrane preparation is boiled along with sample buffer; however room temperature has been also investigated to see if it makes any difference! Samples are then run on 7.5% gel in 90V for at least 2/5 hrs.
It seems proteins are not well separated, specifically proteins with high molecular weight! I only get a smear with almost no detectable protein band. I have conducted the same experiment on different tissues including heart and kidney, but no success do far!
Does anybody have similar experiences? Any suggestion is highly appreciated!
Hey, Aghdas. I don't have much experience. But I have some questions for optimization: Does your RIPA buffer conatin protease inhibitor? Are the procedures done at 4°C continously? Are the centrifugation steps optimal? Did you change the the the acrylamid content in the gel to ensure full seperation? Did you change running conditions? Not detecting a protein is weird, as you apply a huge amount of 75 µg. Is your cell lysis ok? Can you detect the control - GAPDH or else ? Did you try different antibody dilutions? I hope this gives you a better idea of changing parameters. Good luck and best regards!
Hi, some figures could be useful, comassie for example. If you see immediately some problems, during the run, I suppose you did not perform a ransfer step. From your description as just point out can be some dregradation issue, or maybe, you do not resuspend well your protein amount and you have problem in extraction in addition to degradation. When you prepared your samples did you perform a proteins quantification? How is your pellet when you pipetted up and down? Viscous or not ?
I agree with some of what has already been said:
A picture of your gel would be very useful
What exactly do you mean by large proteins versus separation. You need to check the optimal seaparation of your proteins versus % acrylamide:
In general, a gradient gel of 4-12% acrylamide is a good "catch all" for optimal separation of proteins in the range of 20kd to about 100kd
If the proteins you are specifically interested in are at or above 100kd and you are running a single % [email protected]% i would suggest that might be too high and what you need for optimal separation coukd be nearer 4%
But google: there are many and very good tables by companies like Biorad Gibco life sciences etc etc which show % versus size range separation
Also you mention smearing:
How much lysate are you loading in total onto your acrylamide gel? Much more than 40ug per lane for these higher proteins could contribute to this
Finally, old reagents can cause this: if your Temed, APS and acrylamide are more than 1 year old - especially if the Temed and acrylamide having been sitting at room temp - dispose of and buy again
The same goes for pre cast gels that are more than 2 years old
Finally, is your buffer made fresh or from concentrated stock solutions? If the latter, make sure they are no more than 6 months past expiry date
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