I loaded 40ug of protein for each sample. During 15 min of PAGE run, i saw a ''mountain like structures'' only in the wells where i saw vertical straight lines in Ponceau staining.
Some more description:
Gel: Precasted 4-12% Invitrogen Gel (Ref# NW04122BOX)
Gel running buffer: 1x MOPS
Gel Running Voltage: For first 15 min 80 volts and then 130 V for 1 hour.
Western blot conditions: 10V for 75 min in Bis-Tris Buffer with 10% methanol.
When you see these streaked lanes the most likely reasons are too much protein in the lane or the presence of some other solute in the sample which can alter the electrical field, for example too much salt. These are usually the culprits for the narrowing you see of the bands. If you trust 100% in that all of your lanes effectively have 40ug of protein, then you could dialyze the affected samples or exchange buffers by some other method. Or decrease the quantity you seed.
Thank You Sabrina for your quick answer. I will consider measuring the protein concentration of these samples again. Regarding salt problem, all of these samples are kidney lysates made from same RIPA Buffer lot on the same day.
thanks again... Best, Robin
I agree with the others, it seem to be a diffusion because of an imbalance of salt between gel and loading (or running) buffer.
I agree with the others, it seem to be a diffusion because of an imbalance of salt between gel and loading (or running) buffer.
I agree with the others as well. I would not rule out the Salt issue. Even if you used the same RIPA buffer, you may not have put the same quantity of RIPA between differents lanes (depending on your initial protein concnetration in your extract).
Dear all,
Thanks a lot all for your kind replies. I am still struggling to beleive that it can be a salt problem. Here is the additional information for your interest:
1. Lysates prepared in the RIPA buffer
2. Protein concentration was meausred.
3. (Just because i should not thaw the original lysates again for each wetern blot and) Equal volume of all the lysates were taken and mixed with 4x Lämmli dye (For eg: 21ul of lysates and 7ul of 4x Lämmli dye) and then stored at -80.
4. To obtain same protein amount i took different volume of lysates (prepared in step 3) and dissolve in 1x Lämmli dye and load equal volume on to the Gel.
5. Volume of sample from step 3 to Step 4 in lane 9 is atleast same volume or less volume than the samples in lane 2-4. Therefore this gives me feeling that it cant be the problem of salt.
Thank you everybody.
Best, Robin
thanks Ian for your comment. I really dont know if this could be due to the genomic DNA.
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