I am new to western blotting and am practicing the procedure up till the transfer step (I'm good after that step). After the electrophoresis step I take a look at the total protein on the gel, and the attached image is what I get every time; not many bands, and this weird diffusion of the signal into nothing towards the bottom. I've been walked through a western by a very experienced researcher before and the total protein results were very clean and clear, so I know what is possible with my samples.
Can anyone tell me what I might be doing wrong??
Hi Lyle,
do you buy SDS gels or do you prepare them on your own? Which concentrations do you use? How much protein do you load onto the gel/lane?
Kind regards
Kai
Hey Lyle,
Hope you have resolved with your problem by now but if not, I would suggest you to run the gel at around 55volts untill the bands cross the stacking gel, then you can increase the volt to 100. But, remember for better resolution, do not fluctuate the electricity again and again and run the gel upto the end. Shaky bands could also be a resultant of inappropriate buffer, so make sure you prepare the 1X tris glycine buffer freshly and take care that the electrodes are dip in the buffer throughout the run and transfer. Then, while setting up the transfer cassette, remember that the sequence in which you put everything is correct. If using the BioRad cassette, start from the black side (cathode), fibre pad, filter paper, gel, membrane, filter paper, fibre pad. The pads, filters and membrane should be pre-incubated in transfer buffer. Make sure to load a pre-stained marker and run the transfer at 200mA for 1 hr and also place an ice pad and a magnetic bead in the tank and keep it onto a stirrer to dissipate the heat generated. you can also check the transfer by Ponceau staining.
All the very best for your next blot.
Thanks for your answers guys! I haven't been able to test this yet but I found out my lysis buffer had 10% NP-40, and it was only supposed to have 1%. Whoops!
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