hello I run my self made 12 % SDS-PAGE gel for visualization of my extracellular proteins of yeast from media. I take 200 ml of media even but my bands on the gel are less conspicuous. I concentrate my protein using a centrifugal column and run my sample with commercial loading buffer 5X (1:4). brief hearting at 95 for 3 to5 mints, centrifugation for 45 at 12000rpm. i insert all of my 40 ul in a single well.
kindly mark my mistake .
Hello
Since your ladder is not stained properly, there could be a problem of staining/de-staining your gel.
I agree with Ali Javadmanesh .
How old is your staining solution? Coomassie forms aggregates, when it get older. If this happens in your case, they may be too large ot penetrate your gel.
It is difficult to discuss colors in a photo, if it is not calibrated. But if the color of your gel photo agrees with reality, the solution could either be too old or the pH could be off.
Hello Shaz St ,
Do you mean that you concentrate ~200mL media to a few uLs? It might be possible that proteins are precipitating during the process. It would be useful if you estimate the total protein in the sample before running the gel. Then, load different amounts of protein in different wells ranging from 20-50 ug per well, along with a control such as a known conc. of BSA.
And yes, there might be an issue of staining-destaining solution as pointed earlier.
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