This protocol provides a step-by-step procedure for sample collection, extract preparation, and immunoprecipitation for the confirmation of a successful protein pull-down and the detection of co-immunoprecipitated proteins of interest. The protein extract preparation can be performed for up to 24 samples and it's compatible with a number of downstream applications, including immunoprecipitation and RNA pull-down experiments. This method can be adapted to facilitate the testing of interactions between two or more endogenous, endogenously tagged or over-expressed C.elegans proteins in a variety of genetic backgrounds.
Visual demonstration of this protocol is intended to make researchers comfortable with protein extract preparation and immunoprecipitation, hopefully encouraging those new to the techniques to use them in their research. To collect a worm sample, first seed mixed stage or synchronized worms on solid nematode growth medium plates at the required temperature and allow the worms to grow until the desired stage. At the end of the incubation, use M9 buffer to wash the worms into a 15 milliliter conical tube and pellet the worms by centrifugation.
At the end of the centrifugation, remove the supernatant and wash the worms an additional three to five times in fresh M9 buffer per wash. When the supernatant is no longer cloudy, perform one final wash with double distilled water and transfer the loose worm pellet into a 1.5 milliliter microcentrifuge tube. Pellet the worms with an additional centrifugation and discard the remaining supernatant to obtain a packed worm pellet.
To prepare extract from the collected worm pellet, add an equal volume of ice cold 2X lysis buffer to a recommended 300 microliter volume pellet and vigorously mix the resulting suspension. Spin down the sample to collect the mixture at the bottom of the tube and transfer the tube contents into a 1.5 milliliter RNAse-free tube containing metal beads. After capping the tube tightly, place the sample in a bead mill homogenizer at four degrees Celsius taking care that the sample is balanced inside the homogenizer.
Homogenize the sample at the highest speed for four minutes before transferring the sample into a new 1.5 milliliter microcentrifuge tube without beads. Spin down the sample to clarify the protein extract and transfer the supernatant to a new 1.5 milliliter tube on ice without transferring the white cloudy precipitate at the top of the sample. Set aside 10 microliters of the clarified extract to determine the total protein concentration according to standard protocols and immediately dilute the sample to a five or 10 milligram of protein per milliliter of ice cold 1X lysis buffer on ice.
For immunoprecipitation of the protein of interest, first resuspend the magnetic beads by inversion and transfer 150 microliters of the 50X bead suspension into a 1.5 milliliter tube. Magnetize the beads on ice against a magnetic stand for about one minute. When the solution is clear, discard the supernatant.
Remove the tube from the magnetic stand and wash the beads with three 300 microliter volumes of 1X lysis buffer. After the last wash, resuspend the beads in 150 microliters of ice cold lysis buffer and transfer 75 microliters of the bead slurry to two milligrams of the protein extract sample. After a one-hour incubation at four degrees Celsius with gentle agitation, place the sample tube in the magnet on ice for about one minute.
When the beads are fully magnetized and the sample is clear, transfer the supernatant to a new 1.5 milliliter tube without disturbing the beads. Set aside 10%of the sample for Western blot analysis and add 20 micrograms of affinity purified antibody to the pre-cleared lysate for a one-hour incubation at four degrees Celsius with gentle agitation. At the end of the incubation, add the remaining 75 microliters of pre-washed bead suspension to the antibody lysate mixture for an additional one-hour incubation at four degrees Celsius with gentle agitation.
At the end of the incubation, place the sample back onto the magnet for about one minute. When the beads are fully magnetized and the sample is clear, wash the beads containing the immunoprecipitate three times in 450 microliters of wash buffer on ice. After the last wash, resuspend the bead pellet in 20 microliters of 2X SDS beta-mercaptoethanol protein gel loading buffer and boil the sample at 95 degrees Celsius for five minutes.
For detection of the immunoprecipitated proteins by Western blot analysis, load the denatured protein sample onto an SDS-PAGE gel without transferring the beads and perform Western blot analysis according to standard protocols using the appropriate antibodies for the proteins of interest, then detect the bands using horseradish peroxidase-based chemiluminescence. The demonstrated bead mill homogenizer protocol is comparable in total protein extraction to dounce-based methods for the efficient extraction of nuclear and cytoplasmic proteins. In this analysis, Argonaute proteins were determined to interact with members of the GW182 protein family forming the microRNA-induced silencing complexes that bind to the target messenger RNAs and repress their expression.
The demonstrated extract and immunoprecipitation protocols can also be used to successfully recover ALG-1 and HRPK-1 specific co-immunoprecipitates. In addition, testing of the ALG-1:AIN-1 interaction in a variety of genetic backgrounds revealed that HRPK-1 is unnecessary for the ALG-1:AIN-1 microRNA-induced silencing complex assembly. It is important to make sure that protein extraction and the immunoprecipitation are performed at four degrees Celsius or on ice to maintain the stability of the samples.
This total protein extract preparation is compatible with protein immunoprecipitation and microRNA pull-down experiments using a microRNA complimentary oligonucleotide and facilitates the downstream collection of both protein and RNA components.
