problem in running with 2x sample buffer

[MIKAH]

Hej,

I am using 2x sample buffer for running my lysates in SDS page. when i do so , I get a broad blue region below the BFB line (i.e below the sharp bromophenol bue indicator line). Further my BFB line is kind of thick instead of sharp.

Could any one face the same problem?? or any idea to overcome it

p.s my samples becomes very diluted when i go with 1x Sample buffer. also i hear the pH of resolving buffer and Sample buffer has some direct correlation.
recipe of SB is correct.

Help me out if you got any idea

[butters]

Exactly what formula SB do you use?

Do you mean if you put same amount say 5uL of your lysate with 1X (diluted) or 2x(too thick) visible after staining?

Is it possible that you share with us the picture?

[MIKAH]

Hej,

Thanks for bringing up to discussion.

The recipe of 2X SB is the same as we use normally. The pH is around 8. Similarly the pH of my resolving buffer is 8.3 and my stacking buffer is 6.8.
I have attached the image of the gel pic. If you see the pic, u ll find blue broad zone below the BFB line.. Can u figure out why that happens and any idea to rectify it????


P.S. I didnt have any gel pic to send immediately, so late response.