I have tried alot and don't know the reason why these bands are distorted? Can any one suggest me? the pic is attached below.
Its probably your sample+loading dye is concentrated and needs more dilution. OR the wells in the gel needs cleanup.
that means i have to decrease the concentration of the loading dye and also clean up the wells with water or run the gel for 10 min without loading the gel.
It might be due to high concentration of protein in your gel. Don't load too much protein.
Thanks Khadijeh Gholami. Can it be due to high concentration of antibodies also because i increase antibodies than usual. Plz reply.
Hello
The gel may be contaminated , also i suggest you keep running time you completely cool condition and check voltage in properly
find the attachment here
Good Luck
Sumeet, could you specify a little bit about you sample. Is it tissue, cell culture, plants ... . If it is cell culture I would recommend to discard as much media as you can before adding lysis buffer (ions, salt and proteins from media could be a cause of your problem) . For tissue you should find special kit or optimize usual protocol.
centrifuge your sample before loading and clean your comb while making the gel and an run the gel in cold enviromet
Evgenii Boriushkin i am doing in cell culture . And i wanted to ask one question can the high concentration of antibodies can effect like this? plz reply.
Antibodies added for detection of protein on the membrane following transfer do not affect running of the gel prior to this. So no, it is not due to high antibody concentration. CLean out the wells before loading sample (pipette running buffer in and out of each well). MAke sure the sample buffer is diluted to 1 x. Load empty neighbour wells with 1x sample buffer as empty wells can "pull" the sample strangely. Dont run the gel too fast so it gets warm, maybe the cold room helps here as well. Dont overload with protein, your actin bands are blasting, you could reduce the amount of sample loaded
good luck.
I don't think so. It's very easy to check it: stain your gel by coomassie or ponceau staining before transfer (or other simple staining by dye).
Thanks Fergal O'Farrell. But i usual load 4 x loading buffer . is it ok to use 4x loading buffer instead of 1x loading buffer in all sample and also in the empty neighbouring well?Ok i will reduce my protein sample also.Waiting for your reply....
No, you should not load 4x loading buffer on the gel. This would run strangely. Thats like 40% glycerol. When prepping my samples i add 25 ul of loading buffer to 75 ul of cell lysis. (1 to 4, so know the loading buffer is 1X). I then load say 20ul of this per well. Blank lanes on the gel i load with 20ul of 1X loading buffer (e.g. 25 ul of loading buffer mixed with 75ul of water). Theres a lot of glycerol in the loading buffer, IF you dont do this the amount of glycerol will vary between the wells. Again pure 4x loading buffer on the gel will not run nicely. Good luck.
Thanks Fergal O'Farrell. I will try that on my next western blotting. I used to add 25 ul 4 x loading buffer in 75 ul of cell lysis.
Thanks Fergal O'Farrell you were right. its ok now. Thank u very much once again....
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