Protein Concentration Determination

Materials

Motor protein (>95% purity, 15 - 20 µM)

Bio-Rad Protein Assay Reagent

BSA (500 µg/mL)

HEM =

10 mM HEPES, pH 7.2
1 mM EGTA
1 mM MgCl₂
1 mM DTT

Procedure

1.	Set up assay tubes for BSA standards = 0, 1, 2, 4, 8 µg/mL

BSA Concentration	Pentex BSA (500 µg/mL)	HEM
0 µg/mL	0	400 µL
1 µg/mL	0.8 µL	399 µL
2 µg/mL	1.6 µL	398 µL
4 µg/mL	3.2 µL	397 µL
8 µg/mL	6.4 µL	394 µL

2. Dilute motor protein 1:800, 1:500 and 1:400. Centrifuge for 5 min at 4°C prior to assaying.

Protein Dilution	Protein	HEM
1:800	0.5 µL	399 µL
1:500	0.8 µL	399 µL
1:400	1.0 µL	399 µL

3. Dilute Bio-Rad reagent 1:4 (200 µL Reagent + 600 µL DDW = 800 µL)

4. Add 100 µL to each assay tube. Let tubes sit for 3-5 min at room temperature, then read OD₅₉₅ using 0 BSA as blank.

5. Plot BSA concentration vs OD₅₉₅ reading. Determine concentration of motor protein from curve of BSA standards.

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Created 6 November 1999 19:26 GMT
Modified 7 May 2001 18:25 GMT