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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PURIFICATION: EXTRACT FROM CELLS

RAPID PREPARATION OF PROTEIN SAMPLES FROM MAMMALIAN CELLS FOR WESTERN BLOTTING ANALYSIS

Rapid Preparation of Protein Samples from Mammalian Cells for Western Blotting Analysis
Contributor: The Laboratory of Daniel Portnoy at the University of California, Berkeley
 
Procedure
1. Wash cells twice with PBS and aspirate the PBS (see Hint #2).

2. Add 1 ml of PBS to each plate, scrape the adherent cells with a rubber policeman to dislodge the cells into the PBS, and collect the cell suspension in a microcentrifuge tube.

3. Centrifuge cells at approximately 8,000 rpm in a microcentrifuge for 10 min.

4. Aspirate the supernatant and estimate the cell pellet volume (see Hint #3).

5. Add 1 μl of 10X Lysis Buffer plus Protease Inhibitors per 10 μl of cell pellet volume (or add an equal volume of 1X Lysis Buffer with Protease Inhibitors).

6. Vortex for a few seconds to mix well.

7. Add 0.2 volumes of 1X Lysis Buffer/DNase I Solution.

8. Incubate the cell suspension at 37°C for 10 min.

9. After the incubation, add 0.2 volumes of 5X Protein Loading Sample Buffer.

10. Heat the samples in a boiling water bath for 1 to 2 min.

12. Use between 1 μl to 20 μl of samples for Western analysis (see Protocol ID#455 and Protocol ID#1146).

Solutions
Lysis Buffer with Protease Inhibitors   Prepare in 1X Lysis Buffer just before use
1X Leupeptin Stock
1X Pepstatin Stock
1X Aprotinin Stock
1X PMSF Stock
Aprotinin Stock (200X)   10 mg/ml Aprotinin
Store at -20°C
Leupeptin Stock (100X)   1 mg/ml leupeptin
Store at -20°C
Pepstatin Stock (400X)   Prepare in 100% Ethanol
3 mg/ml Pepstatin
Store at -20°C
PMSF Stock (500X)   Prepare in 100% Ethanol
43 mg/ml PMSF (CAUTION! See Hint #1)
Store at -20°C
Lysis Buffer (10X)   30 mM MgCl2
100 mM NaCl
5% (v/v) IGEPAL CA-630 (Sigma, replaces NP-40)
100 mM Tris-HCl, pH 8.0
Store at 4°C
PBS   pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl
Protein Loading Sample Buffer (5X)   5% (w/v) SDS
5% (v/v) 2-Mercaptoethanol
50% (v/v) Glycerol
Store at -20°C
0.02% Bromophenol Blue
Lysis Buffer/DNase I Solution   Prepared in 1X Lysis Buffer
1X DNase I Stock
DNase I Stock (10X)   50% (v/v) Glycerol
Store at -20°C
150 mM NaCl
1 mg/ml DNase I
 
BioReagents and Chemicals
Pepstain
Leupeptin
Glycerol
PMSF
IGEPAL CA-630
Tris
Bromophenol Blue
Potassium Phosphate, Monobasic
Ethanol
2-Mercaptoethanol
Sodium Phosphate, Dibasic
SDS
DNase I
Aprotinin
Potassium Chloride
Sodium Chloride
Magnesium Chloride
 
Protocol Hints
1. This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.

2. Washing conditions will vary depending upon cell type and must be empirically determined.

3. Typically, the cell pellet volume is between 10-50 μl.