I work with protein secretion using condition media. I've collect and concentrate the condition media using 10kdA Amicon Ultra Filter (Milipore) and proceed with SDS page at concentration 4ug/ul to check the availability of protein. However the band look so faded (see picture) . Does anyone know how to solve this problem? And what is the ideal concentration used for SDS?
* the left side is the ladder (25-250bp). The rest are the samples .
You can to precipitate your protein using isoelectric point protocols with ammonium salts. Also, you can lyophilize your sample.
If your purpose is to concentrate the proteins just for analysis by SDS-PAGE, you can use the trichloroacetic acid precipitation method. This allows you to concentrate the total protein content of 1 ml of sample for SDS-PAGE in a single lane. (Add 100 µl of 0.15% sodium deoxycholate and 100 µl of 72% TCA to a 1 ml sample, centrifuge at top speed in a microfuge for 10 minutes, carefully remove all the supernatant, and dissolve the precipitated proteins with SDS-PAGE sample buffer).
For a mini-gel, 20 µg per lane should give you lots of protein to stain with Coomassie Blue. Since your sample had 4 µg/µl, only 5 µl should have been sufficient without further concentration. Either your protein measurement was wrong, or your staining procedure was unsatisfactory.
If you were using medium containing fetal bovine serum, the great majority of the protein in the sample will be bovine serum albumin (BSA, 66 kDa). There are products available for removing the albumin, if needed. Using serum-free medium avoids this problem.
Hi
Maybe this aricle can halp
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2386146/
good luck
If media contain serum, TCA precipitation is not recommended because co-precipitated lipids make the pellet extremely difficult to solubilize. Ammonium sulfate (especially 80%) would affect the electrophoretic mobility of your proteins (due residual salts). Dialysis could be considered to eliminate salts before SDS-PAGE but It would dilute your samples...
Finally I would suggest Methanol-chloroform precipitation. It's a two steps procedure. In the first one, proteins and lipids are separeted in 2 distinct phases (lower phase : proteins, upper phase : lipids). After the removal of the upper phase (chloroform), proteins are precipitated with methanol. This second step allows the precipitation of your proteins and the elimination of lipids and chloroform traces at the same time.
Protocol: https://rtsf.natsci.msu.edu/_rtsf/assets/File/MethanolChloroform.pdf
Thank you for all the suggestions. I've go some reading but kind of confuse on the precipitation method. My condition media contained no serum. Therefore, should i go for TCA percipitation or ammonium sulfate precipitation or Methanol-chloroform precipitation? Does all this method would give any bad effect to my protein samples?
I conduct SDS as to make sure the protein is there and afterward im going to run 2D Gel.
TCA precipitation may not be advisable for 2-D electrophoresis because it may not be possible to dissolve the pellet without adding SDS, which would interfere with IEF.
You can try an other method for dyaing the proteins after gel electrophoresis, i.e. silver dying.
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