Hi everyone !
I am doing western blots on mice skin extracts and tick salivary glands extracts, which I run on a Bio-Rad's TGX Precast gel 12%. I have two bands of interest that show up in both the skin and the salivary glands extracts, but from one blot to the other they are not exactly at the same molecular weight. Precisions : my molecular weight scale is the same, I am using the same extracts every time, putting the same amount of proteins and running the gel for the same time at the same voltage (40min, 200V).
My question is : do I need to change my SDS-Tris-Glycine running buffer for every new run (as recommended by one of our technicians) or can I reuse it 4-5 times (as I'm doing since the beginning) ?
Thanks a lot for all the suggestions, ideas and critics !
If the experiment is not EXTREMELY sensitive to changes just change the buffer once every day, or even less. Changing it more often is more a paranoid act rather than rational. Of course it also depends on the runs you do. If you work (for example) with radiolabeled substrates you can contaminate your gels then you may take care of this but in a regular experiment is just unusual that it makes a big change.
I would recommend using fresh buffer every time at least to avoid contamination with other proteins if you over run your gels.
Good luck
Thank you all for your answers !
Omar, you say changing the buffer once a day is sufficient but how many runs do you do in a day ?
Ali, as I said I'm using Bio-Rad's TGX Precast gels, I only need to take them out of their box and put put them in the tank so ... already polymerized.
Aamal, that is exactly what I'm going to do now even if I don't usually over run my gels (I stay in font of my tank the whole time ...).
Again, thanks everyone !
I have seen labs where they run like 6-8 gels per day with the same buffer with no problem.
Ok thank ou ! I used to run 4-5 gels in the same buffer but not the same day ... Does it change anything ? (conservation of the buffer, alteration with time or something ?)
Dear Giroudot,
Excellent suggestions shared already. Just wanted to add although there will be ionic imbalance after you run an electrophoresis as already ionized molecules will stick to the electrode and won't participate in the mobility. So, effectively there will be lesser ions available after each subsequent run and the elecetrophoresis might slow down due to gradual deprivation of ions.Thus, reusing should be restricted to an extent. But that should not be playing pivotal role in changing the mobility of proteins in a way to not coincide with expected molecular weight marker. Make sure your marker is running properly and you are not picking up any non specific band. It happens at times that the proteins do not match exactly the expected size according tot the molecular weight marker, but the difference should be nominal. As suggested, you should use some other proteins which have worked on earlier and see if you experience similar shifts in mobility. I reuse the lower tank buffer only once, but take fresh buffer for the upper tank every time. Also, wash off the gel tank and electrodes properly to avoid too much accumulation of salts that might interfere with the running.
Hope it helps!
Thanks everybody !
I always keep a constant voltage through the entire run, and my molecular weight marker seems fine (no distorted or altered bands). And for the buffer, my gels are prepared in a plastic cassette that includes the reservoir for top (-) buffer so I have no choice but to change it for every new run.
All the information you gave me will help a lot, again thank you so much everybody !
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