treatment buffer concentration - (Jun/09/2008 )

hi there!
i just need a confirmation whether treatment buffer concentration will effect the actual protein concentration that we load into gel? a little bit confusing since i need to load specific amount of protein in each well.
the treatment buffer (2X) mixture are consist of
2 ml glycerol
1.25 ml 1M Tris/Base pH 6.8
4 ml 10% SDS
1 ml B-merchapto
and few grains of bromophenol blue.
the buffer is diluted to 1X prior to use...

thank you...

Our recipe is a little bit different (sucrose instead of glycerol) but the principle is the same. We use a 3x stock and add the ß-Mercaptoethanol (900 µL 3x + 100 µL ß-ME) just before using (it evaporates, so you can't store the buffer with ß-ME very long). If you dilute the sample buffer I guess you dilute it with your sample. You may get problems with the calculation if your protein concentration is very low, then it may be more appropriate to use a 3x buffer.

hi there!
i just need a confirmation whether treatment buffer concentration will effect the actual protein concentration that we load into gel? a little bit confusing since i need to load specific amount of protein in each well.
the treatment buffer (2X) mixture are consist of
2 ml glycerol
1.25 ml 1M Tris/Base pH 6.8
4 ml 10% SDS
1 ml B-merchapto
and few grains of bromophenol blue.
the buffer is diluted to 1X prior to use...

thank you...

thank you for the suggestion and help..i had continue with my recipies, and after transfer to membrane, i got the band of the proteins..so, it seems that, there are no effects of treatment buffer on protein concentration..perhaps......

I make 2X and never dilute to make 1X...

I use 20 ul of 2x + 20 ul of lysate. heat for 5 min at 95 degrees. add 20ul in Biorad apparatus wells.