Hi Carly Wiersma

The amount of protein you have loaded is too much! Again, there can be multiple reasons as why you are not seeing any gaps. It may be because during pipetting your sample in the well, it might have over-filled the well and leaked to next. Secondly, when you are trying to keep a blank well in between samples, try to load some 1x sample buffer in the well rather than leaving blank. It will prevent peak broadening. Are you trying to do Western blot? If yes, try to use multiple X-ray films to capture the signal which might give a good blot!

Hope this helps!

Varsha

Try reducing your protein to 10-15 ug/well. Histone H3 is a highly abundant molecule, this is why you get such image

hi

you can do :

1- Run one SDS page 15% by gradient cell lysate concentration

( 20ug/well,40,60,...170) . normally 20ug/well is ok .

2- you can used from HeLa cells, treatment with sodium butyrate as positive control in your page

3- Instead of increasing the concentration of lysate, increase the incubation time with the primary antibody ( over night at 4 c) or increase the concentration of primary antibody .

4- If the problem is not resolved, you can use the IP(Immunoprecipitation)method .

good luck

Thank you everyone for your advice. I diluted my samples 1:10 and loaded different amounts of protein (150ug, 100ug, 80ug, 60ug, 40ug, and 20ug) and it is very obvious that the amount of protein was the issue. I can clearly see how much the 150ug band is spreading out to fill the lanes next to it compared to the 20ug lane. Problem solved!

Because performance was not adequate!