METHOD 1 –96 Well Plate
Modified Lowry Protein Assay Kit (contents: Modified Lowry Protein Assay Reagent, Folin &
Ciocalteu’s Phenol Reagent, Albumin Standard, ampules) (From Kit: Thermo Scientific cat# 23240)
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2 mL test tubes (10)
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Elution buffer
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Autoclaved water
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COSTAR (#3595)96 well plate and cover
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Multi-channel pipette
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Spectrophotometer
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Materials
Methods
Use one Albumin Standard (2.0 mg/mL) as stock and use Elution Buffer as Diluent
1.
NOTE: Do not use Sarstedt Plates
Prepare the following BSA Standards Accordingly. Store in -20°.
Vial (name)
Volume of Diluent
Volume and Source of BSA
Final BSA Concentration
A
125 µL
375 µL of stock
1,500 µg/mL
B
312.5 µL
312.5 µL of stock
1,000 µg/mL
C
155 µL
155 µL of vial A dilution
750 µg/mL
D
312.5 µL
312.5 µL of vial B dilution
500 µg/mL
E
312.5 µL
312.5 µL of vial D dilution
250 µg/mL
F
312.5 µL
312.5 µL of vial E dilution
125 µg/mL
G
400 µL
100 µL of vial F dilution
25 µg/mL
H
400 µL
100 µL of vial G dilution
5 µg/mL
I
400 µL
100 µL of vial H dilution
1 µg/mL
J
500 µL
0
0 µg/mL = blank
Prepare the Folin-Ciocalteu Reagent by diluting the 2X supplied reagent 1:1 with autoclaved
water. Each test replicate requires 20 µL of 1X Folin-Ciocalteu Reagent when used with a 96-well
plate. The diluted reagent is unstable and must be made on the same day as use
2.
Pipette 40 µL of each standard and unknown sample into a separate well
3.
Add 200 µL of Modified Lowry reagent to each well at nearly the same moment using a multi-
channel pipette
4.
Immediately mix microplate with spectrophotometer for 30 seconds
5.
Cover the plate with plastic lid and incubate at room temperature for exactly ten minutes
6.
Add 20 µL of 1X Folin-Ciocalteu Reagent to each well using a multi-channel pipette. Immediately
mix with spectrophotometer for 30 seconds
7.
Cover plate with plastic lid and incubate at room temperature for 30 minutes
8.
Measure the absorbance at 750 nm on a plate reader. Subtract the average 750 nm absorbance
value on the Blank standard replicates from the 750 nm value of the other individual standards
and unknown sample replicates
9.
Prepare a standard curve by plotting the average Blank-corrected 750 nm values for each BSA
10.
Lowry Protein Assay
Tuesday, June 19, 2012
3:48 PM
Methods Page 1
