The sample preparation does not require sonication or any other mechanical method to crush the nematodes. Synchronized adult animals first are cleaned by transferring to a NGM plate with no bacteria. Next, 15 to 50 nematodes are picked and placed into 20 μL of DDH2O that is contained in the cup-like part of a severed Eppendorf tube lid (Fig.1). Once the nematodes are released in the water droplet, the lid is put on an intact Eppendorf tube containing 20 μL of 2X sample buffer already pipetted at the bottom of the tube (Fig. 1). The nematodes are spun into the 2X sample buffer (see below) for one minute at 12,000 RPM. The pelleted sample with nematodes is then flash frozen in an ethanol/dry ice bath; at this point the frozen sample can be stored in a -800C freezer until one is ready to run the gel. The volumes used in the protocol can be adjusted to match the sample size (for example, 10μL H2O+10 μL 2X sample buffer for 15 adult day1 nematodes).
Prior to running the samples in a mini gel, the samples are incubated at 950C in a heating block for five minutes, followed by five minutes of cooling at room temperature. The samples are then heated again for the second time at 950C for five minutes and can be loaded onto a gel. The subsequent immunoblot protocol varies depending on the particular antigen and antibody used. In our laboratory, proteins in the gels are transferred to a PVDF (BioRad Inc.) filter using NuPage transfer buffer with 10% Methanol and 0.01% SDS at 20 volts overnight or a shorter period (3 to 5 hrs) until no marker protein is left in the gel. Incubation with the primary antibody is commonly performed in PBS containing 4% blotting grade milk with 0.025% Tween-20 for two to twenty four hours in a cold room (40C), depending on the antigen affinity of the antibody. Incubation with the secondary antibodies is done for two hours at room temperature with three subsequent washes (5 min X 3) with PBS containing Tween-20 prior to treating with ECL solution.
2X Sample buffer: 100mM Na-Tricine pH 7.8, 100mM DTT, 14% W/V Glycerol, 4% LDS, 0.05% CHAPS and 0.002% Bromphenol Blue (can be added later if protein concentration is to be determined for the lysates). The sample buffer can be frozen in aliquots in -200C for long-term storage.
2μL HALT (Protease inhibitors cocktail, Sigma) should be added to 98μL 2X Sample buffer prior to mixing with nematodes.
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