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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PURIFICATION: EXTRACT FROM CELLS

PROTEIN GEL EXTRACTS FROM WORMS FOR ANALYSIS BY SDS-PAGE

Protein Gel Extracts from Worms for Analysis by SDS-PAGE Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Contributor:	The Laboratory of Michael Koelle at Yale University. URL: Lab Web Site
Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
This protocol describes the preparation of protein from whole C. elegans animals for further analysis by SDS-PAGE.
Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
A. Collecting worms 1. Grow up 3 to 5 large (10-cm) plates of worms. This should give approximately 200 μl of packed worms after the washes described below. 2. When the worms have just starved, wash the worms off the plate by putting a little M9 Solution on the plate, swirling gently, and aspirating the liquid off with a Pasteur pipet and collecting the worms into a conical tube. 3. Centrifuge in a clinical centrifuge or equivalent at approximately 1000 X g for 2 min to pellet the worms. Discard the supernatant. 4. Wash the worms once with a few ml of M9 Solution. Repeat Step #3. 5. Transfer the worms in a small amount of liquid to a microcentrifuge tube. Centrifuge briefly at 3,000 X g and remove as much liquid as possible from the pellet. 5. At this point the worms may be frozen at -80°C. B. Embryos and Adult Worms 1. Add Sample Buffer to the worms in the microcentrifuge tube to a total volume of 1 ml. 2. Using a probe sonicator with a microtip, sonicate for 20 sec while the tube is in a beaker of icewater (to keep the suspension cold). Try to prevent any froth from appearing during the sonication. 3. Centrifuge the microcentrifuge tube for 10 min at maximum speed in a microcentrifuge to pellet the debris. 4. Transfer the supernatant to a new screw-top tube (see Hint #2) and continue to Section D. C. Adult Worms Only 1. Add Sample Buffer to the worms in a screwtop tube to a total volume of 1 ml. 2. Boil the sample for 5 min. 3. Centrifuge the sample for 10 min at maximum speed in a microcentrifuge to pellet the debris. 4. Transfer the supernatant to a new screw-top tube (see Hint #2) and continue to Section D. D. Loading the Samples 1. Approximately 30 μl may be loaded on to a 1.5 mm thick protein gel using 5 mm wide sample wells. This is the maximum amount of protein that will run well without streaking (see Hint #3).
Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Sample Buffer    15% (v/v) Glycerol 100 mM Tris, pH 6.8 See Hint #4 Store in a tightly sealed container to keep the 2-Mercaptoethanol from dissipating. 5% (v/v) 2-Mercaptoethanol Optional: 4 M Urea may be added to increase the denaturing effect on proteins. 2% (w/v) SDS Add enough Bromophenol Blue to visualize the dye on a gel M9 Solution    22 mM Potassium Phosphate Monobasic (KH2PO4) 19 mM NH4Cl 48 mM Sodium Phosphate Dibasic (Na2HPO4) 9 mM NaCl
BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Ammonium Chloride Urea Potassium Phosphate Tris 2-Mercaptoethanol Sodium Phosphate, Dibasic Sodium Chloride Bromophenol Blue Glycerol SDS
Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions. 2. Store the sample at -80°C. The samples will degrade when stored at 4°C for a few months. 3. To assure even loading of several samples you should load dilutions of your samples on a gel. Then Coomassie stain the gel to determine what volumes of your various samples contain equivalent amounts of protein (see Protocol on Coomassie Staining). 4. More 2-Mercaptoethanol may be needed if the solution is old (as the Mercaptoethanol is highly volatile). Another method is keep the Sample Buffer without Mercaptoethanol and add the Mercaptoethanol just before use.