This protocol makes it possible to label proteins in a cell type specific manner. This is the first technique that offers such a possibility and it can be done in vitro or in vivo. The main advantage of the technique is that the labeled cell type specific proteins can be purified unidentified, or visualized in situ by immunofluorescent.
To begin prepare the tissue lysate by adding lysis buffer at a volume of 12 to 15 times the wet weight of the tissue and triturate the tissue until it is homogenized. Heat this homogenate for 15 minutes to 75 degrees Celsius to denature the protein then centrifuge the sample and transfer the supernatant to a new tube. This sample can be stored at minus 80 degrees Celsius.
Next, for alkylation dilute the homogenized sample two to three times in phosphate saline buffer containing an EDTA-free protease inhibitor. Then add freshly prepared iodoacetamide to a final concentration of 20 millimoles. Leave the sample for one to two hours at 20 degrees Celsius in the dark.
Repeat the iodoacetamide addition and incubation steps twice. Prepare the column as per the manufacturer's instructions by first vortexing the columns. Then removing the lid cutting the bottom part and centrifuging them.
Next, perform a buffer exchange by first equilibrating the buffer exchange columns using the click chemistry exchange buffer and then exchanging all alkylated samples. To evaluate the azidonorleucine incorporation take 40 microliters of the alluded sample and add phosphate saline buffer to a final volume of 120 microliters. Then set up the click chemistry reaction by vortexing the reaction for 20 seconds.
After adding each reagent in the indicated order as fast as possible, incubate the samples in the dark at four degrees Celsius overnight with continuous rotation. The following day centrifuge the samples for five minutes at 17, 000 times G after centrifugation a slightly turquoise pellet will be visible. Transfer the supernatant to a new tube and store the sample at minus 80 degrees Celsius.
For labeled protein purification. Subject all the samples to a buffer exchange procedure as demonstrated to clean leftovers of free alkyne using neutravidin binding buffer as the equilibration buffer. Then wash the neutravidin high capacity beads three times by mixing the beads with the binding buffer.
Centrifuge the mixture, discard the supernatant and repeat this three times. Then prepare a one-to-one slurry by adding the same volume of neutravidin dry beads and neutravidin binding buffer. Set aside 20 to 40 microliters of each sample as pre-purified lysate and store it at minus 20 degrees Celsius.
After measuring the protein concentration of the sample mix one milligram of protein with 40 microliters of the beads slurry. Incubate the mixture overnight at four degrees Celsius with continuous rotation enabling the labeled proteins to bind to the beads. On the following day collect the supernatant by centrifugation.
Set a 20 to 40 microliter aliquot of the supernatant aside for later analysis and freeze the rest at minus 80 degrees Celsius. Next, wash the beads three times by adding chilled neutravidin washing buffer one. Sedimenting the beads and discarding the supernatant.
Then add the same buffer and incubate the beads for 10 minutes under continuous rotation at four degrees Celsius before discarding the supernatant. Repeat the wash with neutravidin washing buffers two and three, and elute the clicked proteins by incubating the beads for 30 minutes at 20 degrees Celsius with a volume of neutravidin elution buffer, corresponding to one volume of the dry beads used. During the elution, maintain the beads in suspension at 1000 revolutions per minute in a thermo block shaker elude twice and combine both elutes.
Click reactions were analyzed by SDS page and Western immuno blot. Representative images of the experiments are shown for azidonorleucine administration by intra peritoneal injection and for azidonorleucine administration via drinking water. A further experiment provides an example for the determination of the optimal disulphide biotin cleavable alkyne concentration.
In this example, the fold change between the labeled sample and control is highest at 14 Micromolar alkyne concentration. An example of a mass spectrometry result with a clear enrichment in the azidonorleucine labeled sample compared to the control is shown here. This difference was already visible in the total protein stain.
Besides changes in peptide intensity there are also unique proteins found in both samples. This is a technically complex protocol and its a step must be carefully followed using proper negative controls alkylation, proper(indistinct)and adequate cleaning of the non click archive. Our key steps in this protocol, purified proteins can be either identified by mass spectrometry if the researchers interest is to study proteins or load it into a gel for studying proteins of interest by western blot.
The ability of this method to label proteins from a specific cellular origin has a lot of applications such as the tension of proteins or the study of protein synthesis in vitro or in vivo.
