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Protein Sample Preparation - SDS Samples
I.
Homogenization Buffer (Crude Homogenates from Whole Heart)
Stock Solution
Amt for stock
Amt for 10ml
3.2M Sucrose
200 mM Tris-Cl
pH 7.4
200mM EDTA
1 M EGTA
100% BME
10mg/ml PMSF
20mg/ml Leupeptin
109g/100 ml
(1ml) 1:10 dilution
II.
2.42g/100ml
3.36g/50ml
19.0g/50 ml
Final Concentration
(1ml) 1:10 dilution
(100l) 1:100 dilution
(100l) 1:100 dilution
100l
10mg/ml isopropanol 5.2l
20mg/ml isopropanol (10 l) 1:1000 dilution
0.32M
20 mM
2 mM
10 mM
0.3mM
20g/ml
SDS Sample Buffer
Glycerol
BME
SDS (10%)
Tris-HCl; 1M pH6.7
Bromophenol Blue
1 ml
0.5 ml
3.0 ml
1.25 ml
1-2 mg
Aliquot buffer and freeze (due to BME being added).
III.
Homogenization and Protein Measurement
Homogenize frozen tissue in approximately 2 ml of buffer per gram of rat tissue. Mouse
tissue varies but approximately 1:25 dilution – see Notes
For Fractionation:
1.
Centrifuge sample at 10,000 g (40,000 rpm for T-50) for 1 hour at 4°C. Save
supernatant as cytosolic fraction – place on ice.
2.
Resuspend pellet in similar (or less) volume of homogenization buffer BUT
without the sucrose and with 1% Triton-X. (Sonicate)
3.
Shake triton mixture for 1hr at 4°C or longer.
4.
Centrifuge sample at 40,000 RPM for 1 hour at 4°C. Save supernatant this is the
particulate fraction – place on ice.
Last Updated: 02/12/16 5:22 AM
D:\116098679.doc
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Protein Measurement (Bradford Assay):
Measure protein concentration in 30l (of 1:20) with BioRAD Assay Mixture (3 ml total
volume). For mouse heart it may be necessary to increase sample volume to 20ul to
bring into linear range of standard curve (Abs between 0.2 and 0.5).
Linear relationship – Abs @595 vs. Protein concentration (g).
y= 14.1x - 0.97
Calculate total protein concentration in samples and volume for 200 ug of protein for rat
tissues and 800-1200 ug for mouse heart.
*Tris interferes with this
III.
SDS Samples
1.
Take desired g volume from stock homogenate and add 2.5-10% TCA to bring
the total volume desired.
2.
Spin at 3000 RPM for 15min
3.
Decant and discard supernatant. Air dry pellet for 30-60 minutes (should be white
precipitate).
4.
Add 200 l of SDS stock, vortex and make sure pellet is dissolved (use approx 68ug/ul for mouse hearts). If sample is yellow add a drop of concentrated NaOH to
titrate color to blue.
5.
Boil samples for 5 minutes.
6.
Cool samples to room temperature and freeze at –20C in Eppendorf tubes.
Notes:
1.
Heart samples obtained from mice should be diluted 1:25 with homogenization
buffer.
2.
Proteins should be precipitated with 2.5% TCA. Add SDS buffer for a final
concentration of 6-8 g/ul. When SDS buffer is added it almost always requires
titration with 1N NaOH – do this immediately and use only 1ul at a time.
3.
Mouse heart samples for Western blot analysis should be run at 60-80 g of
protein.
Last Updated: 02/12/16 5:22 AM