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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PURIFICATION: EXTRACT FROM CELLS

ISOLATION AND EXTRACTION OF PROTEIN FROM MAMMARY EPITHELIAL CELLS GROWN IN 3D ENGELBREATH-HOLM-SWARM (EHS) EXTRACELLULAR MATRIX CULTURES

Isolation and Extraction of Protein from Mammary Epithelial Cells Grown in 3D Engelbreth-Holm-Swarm (EHS) Extracellular Matrix Cultures
 
Overview
This protocol describes the isolation of mammary epithelial cells grown in 3D culture with Engelbreth-Holm-Swarm (EHS) mouse sarcoma extracellular matrix and the lysis of these cells for future protein analysis.
 
Procedure
1. Remove medium from epithelial cells in EHS culture and wash once with cold PBS.

2. Add ice-cold PBS/EDTA Solution and disrupt the EHS matrix with a pipettor. Use 1 to 2 ml at a time for a total of 8 to 10 ml of PBS/EDTA Solution for each 35 mm dish.

3. Transfer the cell suspension to a 15 ml plastic conical tube on ice.

4. Incubate the cells on ice in a cold room on a rocking platform for 1 to 1.5 hr.

5. Centrifuge the cells at 800 to 1,000 X g for 5 min at 4°C in a table-top centrifuge or equivalent.

6. Wash the cell pellet by resuspending the cells in 10 ml of ice-cold PBS/EDTA Solution and centrifuging at 800 to 1,000 X g for 5 min at 4°C. Repeat the wash one more time or until the cell pellet is clear of EHS matrix.

7. Resuspend the cell pellet in approximately 500 μl of ice-cold RIPA Buffer

8. Disrupt the cell pellet by sonication with two 5 sec bursts.

9. Centrifuge the lysate for 10 min at maximum speed in a microcentrifuge.

10. Transfer the supernatant to a fresh tube, aliquot into smaller aliquots and quick-freeze in a Dry Ice-Ethanol bath.

11. Store the cell lysate aliquots at -80°C.

Solutions
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
RIPA Buffer   Protease Inhibitor Cocktail Set 1
0.5% (w/v) Deoxycholate
50 mM Tris-Cl, pH 7.4
150 mM NaCl
0.2% (w/v) SDS
1% (v/v) Igepal CA-630
PBS/EDTA Solution   1 mm Sodium Orthovanadate (CAUTION! see Hint #1)
5 mM EDTA
Prepare in PBS
Protease Inhibitor Cocktail Set 1 (Calbiochem) (CAUTION! see Hint #1)
1 mM NaF (CAUTION! see Hint #1)
 
BioReagents and Chemicals
Deoxycholate
SDS
Ethanol
Tris
Potassium Phosphate, Monobasic
EDTA
Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
Sodium Fluoride
Dry Ice
Protease Inhibitor Cocktail Set 1
Sodium Orthovanadate
IGEPAL CA-630
 
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.