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protein concentration

Elizabeth Harris chlamy at ACPUB.DUKE.EDU
Mon Jun 19 11:09:06 EST 1995


>Does anyone have a good method for measuring total protein concentration in
>a sample of Chlamy whole cells ?

Old-fashioned, but we used to do it a lot -

Count cells in your culture.

Pellet a 10 ml sample at low speed.

Extract with 80% acetone by incubating on ice for 15-20 minutes, vortexing
occasionally, then spin out pellet (you can measure the chlorophyll content
on the supernatant if you want).

Dissolve the pellet in 1.0 ml 0.2 N NaOH - incubate 20 minutes at room
temperature; if it doesn't dissolve all the way, you can heat the tube
briefly in a bath of boiling water.

Assay 0.1 to 0.5 ml of the sample using the Lowry method:

reagents:

3% sodium carbonate (120 g NacCO3 plus 16 g NaOH in 4 liters H2O)

4% sodium potassium tartrate (5.2 g NaK tartrate in 100 ml H2O)

1.28% copper sulfate (2 g CuSO4-5H2O) in 100 ml)


These solutions are stable at room temperature.  On the day of assay, mix
96 ml 3% sodium carbonate, 2 ml 4% sodium potassium tartrate, and 2 ml
1.28% copper sulfate.

Add 2.5 ml this solution to each reaction tube, and add up to 0.5 ml
protein solution, plus water if needed, to 3.0 ml total volume.

Let stand 10 minutes at room temperature.

Dilute 2 N Folin phenol reagent (purchased from chemical supply co.) 1:1
with water, immediately before use, and add 0.25 ml to each reaction tube.

Let stand 10 minutes at room temperature.

Read OD at 660 nm vs. reagent blank, using BSA or another standard protein
solution as a reference to calculate the concentration in your cell sample.



Elizabeth Harris
chlamy at acpub.duke.edu






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