Microsome protein concentration - (Apr/18/2008 )

I can really use some help.

We have prepared microsomes from seed (mature and inmature) and are having problems quantitating the protein concentration accurately and consistent.

I have tried Bradford with addition of 0.1% SDS, the calculations are not uniform across samples and appear to be over estimated (ran Xug on gel and appear lighter than they should).

I have tried BCA assay with addtion of 2% SDS, the concentration values are higher than the Bradford and again not consistent across samples.

I can really use some tips on how you have accurately calculated protein concentration from microsomes. Any information will be a huge help.

-Isabel in Davis

-izzybusydizzy-

QUOTE (izzybusydizzy @ Apr 18 2008, 04:56 PM)

for membranes, total lysates and microsomes we use the Stoschek-modified Bradford protocol which means adding 0.1 M NaOH

-The Bearer-

"for membranes, total lysates and microsomes we use the Stoschek-modified Bradford protocol which means adding 0.1 M NaOH"

I wanted to follow up on this.

I tried the modified Bradford with .1M NaOH and it did not work on my samples, however I decided to use 0.1M NaOH with the BCA assay and it worked beautifully. Thank you for your help in getting to the right direction. I am glad to have this working, I hope this helps others as well.

-Izzy

-izzybusydizzy-

QUOTE (izzybusydizzy @ May 21 2008, 11:07 AM)

nice to get your feed-back;

I wonder why it did not work with Bradford; it is important to add also 0.1 M to the reference proteins for calibration the system

-The Bearer-

QUOTE (izzybusydizzy @ May 21 2008, 11:07 AM)

nice to get your feed-back;

I wonder why it did not work with Bradford; it is important to add also 0.1 M to the reference proteins for calibration the system but I think you have done this

-The Bearer-