Pre-Western Blot -- BCA Assay

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From the pages of my lab notebook: Thursday, December 19, 2013

Purpose of western blot: to check for transgene expression; expression of the NTRK1 or NTRK2 gene in the BaF3 cells (side note: I have an answer to the why model human leukemia in mouse question, to be found in my research paper)

Last week: lysed the cells with cell lysis buffer 

Goal for today: to do a bovine serum albumin (BSA) protein assay--with BCA (bicinchoninic acid), a chemical reagent that colors the protein
Using known concentrations of BSA protein, we can make a standard curve based on optical density (OD) and measure an unknown protein concentration against the curve. This is a colorimetric assay (use a spectrophotometer).

Test tube procedure:
-I make up different concentrations of proteins by diluting with a diluent (1X lysis buffer) 
-then, I calculate how much working reagent I need (a mix of sample buffer, which is blue, and BME, a stinky chemical) to denature the protein from its 3D form into a linear form
-mix proper amounts of working reagent with protein
-incubate at 37 degrees C
-color should change (more protein=more purple)

At the spectrophotometer:
-Turn visible light on
-Read at 595nm
-Note standard concentrations at ug/mL

-Create standard curve
-Start reading "unknown" samples--NTRK1 and NTRK2 lysates (mutations and WT)

Data in my lab notebook--which I can show to you after break. (All samples have protein expression). 


Comments

I look forward to seeing the

I look forward to seeing the lab notebook and the Western images! Now that we're officially approaching 2014 and time to do experiments is growing sparse, perhaps a worthy topic to write about in a future post would be what experiments you have finished vs. what experiments you would still like to do in the remaining time.