Page 1 of 3
UNIVERSITY OF LEICESTER
CANCER STUDIES & MOLECULAR MEDICINE
Cell Signalling Section
STANDARD OPERATING PROCEDURE
SOP - 607
TITLE:
VERSION - 01
Protein Quantification
Written by: Angie Gillies
Reviewed by: Lindsay Primrose
Changes introduced:
Implementation date: 19.06.08
Review date:
19.06.10
1. PURPOSE To provide a method for the quantification of protein in a sample.
Note: Often referred to as a Bradford Assay.
2. SPECIAL NOTES
Full details of the use of this procedure must be recorded in the appropriate laboratory
notebook, in accordance with SOP 105.
3. CROSS REFERENCES
3.1
3.2
3.3
Preparation of protein lysates
Preparation of conditioned media for Western Blotting
Protein quantification
SOP 605
SOP 606
CPA 093
4. EQUIPMENT & MATERIALS
4.1
4.2
4.3
4.4
Protein Assay Dye Reagent Concentrate
Bovine Serum Albumin
Sterile ultrapure water
Spectrophotometer
4.5
Gold Lysis buffer (stored at room temp)
Sops\sop607
Bio-Rad 500 0006
Sigma A-3294
RPM 23
ThermoSpectronic
Genesys 10uv
RPM 114
Page 2 of 3
5. PROTOCOL
5.1
Prepare Dye Reagent by diluting 1:5 in UP H2O (2ml Dye + 8ml water). Store
at 4°C in foil-wrapped tube
Diluted Dye Reagent can be used for up to 2 weeks.
Ideally a standard curve should be constructed each time an assay is performed
but, at the very least, one should be made each time a fresh dilution of Dye
Reagent is made.
Construction of Standard Curve:Prepare a stock BSA solution (BSA in UP H2O) as accurately as possible at
10mg/ml (i.e.10µg/µl). Aliquots can be stored frozen at -20°C.
Now prepare a working stock solution by diluting the stock BSA solution (A)
by a factor of 1:100 (10µl Solution A + 990µl H2O). The concentration of this
solution is 0.1 µg/µl.
Using the 0.1 µg/µl working stock solution, make the following dilutions for the
5.2
5.3
5.4
5.5
5.6
standard curve:
Vol of BSA (l)
Vol of water (l)
Dye reagent
0
5
10
20
30
40
60
80
800
795
790
780
770
760
740
720
200l
200l
200l
200l
200l
200l
200l
200l
Protein
concentration(g)
0
0.5
1
2
3
4
6
8
Note: we have found that anything above 8µg protein falls outside the linear range
5.7
5.8
5.9
5.10
5.11
5.12
Vortex the samples.
Store in the dark for 10 minutes.
Read the absorbance at 595nm with the white lamp, using disposable 1ml
cuvettes. Use the first sample (0µg BSA) to blank the spectrophotometer.
Construct a standard curve in excel and apply trend line. Use the equation of
the trend line to calculate protein concentrations from absorbance readings.
Absorbance readings of the samples MUST fall within the range of the
standards. For this reason it may be necessary to prepare 1:10 dilutions of the
samples as well as measuring them ‘neat’.
Quantification of Samples:Prepare samples and relevant blanks as follows:
For samples



795µl UP H2O
5µl sample
200µl diluted Dye reagent
For cell lysate blank: Use 5µl Gold Lysis buffer instead of sample
For Conditioned media blank: Use 5µl (any) depleted media instead of sample
Sops\sop607
Page 3 of 3
5.13
5.14
5.15
5.16
Proceed as for the standard curve samples.
Use the trend line equation obtained from the standard curve, to calculate the
concentration (µg) of protein in the 5µl sample.
From this, calculate the concentration of protein per µl and hence the volume
of sample required for 25µg protein.
25µg of protein per well is usually loaded on a Western gel. Occasionally if
the expression of the target protein is low, more can be loaded.
Sops\sop607