gel staining, help

[aoi]

hello all.

im doing the 2D gel electrophoresis. i used silver staining method but somehow my gels turn out to be very dark in colour (making the spots hard to see). can anyone gives opinion/suggestion on why this happened? ty

[Jyona]

hey which silver stain kit are u using? Generally kit comes with detail instructions and most important step is adding stain and then developer with enhancer. The background gets to dark if you over stain the gel. You can try loading more sample in each compartment 1-2 ugm and see if that rectifies the problem

[aoi]

i didnt use staining kit but conventional method instead. im thinking of reducing the developping time..

[Jyona]

ideally the developing time should be 2-3 mins and can take a min more if you are looking for even fainter bands......but more than that do overstains the background so if you generally load lil more of sample the bands develop darker quite soon n in this way you are not overstaining your gel. You can make samples in (1:2 or 1:3) ratio i.e. one part 2x loading buffer and 2/3 parts of your sample.

[aoi]

thankz a lot for the suggestion

[tomoe]

agree Jyona

for developer solution --> 4 C.
use clean gloves (non powder)

[laxii]

hi every body;
I need your help plz. I am new in using WB but I am using it now for 3 months and I could not get expected signal for the protien of interest. I am folloing the protocol exctelly and I tried to optomize it by different ways like increasing transferring time and blotting time, using different gel percentage, using other's control samples etc. the protien Mol. Wt. is 86-90 KD. please help me I am wasting the time and my supervisor does not happy!