A simple, quantitative immunoblot protocol using equal numbers of nematodes

We have developed a protocol that greatly speeds preparation of C. elegans samples for analysis by immunoblotting. A key point of simplification in the protocol is standardizing the age and numbers of nematodes used for the studies. Our immunoblot analyses use 15 to 50 age-synchronized nematodes per well of 10 or 15 lane mini protein gels (Invitrogen). The exact number varies depending on the nematode age, the antigen abundance and the quality of the antibody. However, to provide investigators a benchmark from which they can further optimize their studies, we provide three examples using Actin as the antigen and hermaphrodites as the source material: L4/35 nematodes, adult day1/30 nematodes, adult day3/20 nematodes. Use of a defined number of age synchronized nematodes enables us to load exactly the same amounts of total protein from multiple samples with very minimum variation between the gel wells.

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.

Figures

Figure 1: Schematic details of transferring nematodes to the lids of an Eppendorf tube.

Figure 2: A 4-20% gradient gel with total sample volume of 20 μL containing 15 nematodes. β-Actin (mouse, MAB 1501, Millipore) bands are shown at ten seconds exposure.