Immunoprecipitation with an Anti-Epitope Tag Affinity Gel to Study Protein-Protein Interactions

The scope of our research encompasses exploring the molecular mechanisms that regulate the development and function of brown and beige fat. Trunks, fiction factors and co-factors, such as the PRDM 16 complex, NAFRA, and PPR alpha, ELK one complex that we have more recently found. Control brown and beige fats have faded in function.

In addition, immune cells adipocyte crosstalk have been identified as key players. To advance research in your field, we employ several cutting edge technologies. These include quantitative PCR and chromatin immunoprecipitation for analyzing changes in gene expression, as well as called immunoprecipitation and mass spectrometry to investigate alterations in protein protein interactions, PPIs, associated with differentiation into beige adipocytes.

Despite following the standard protocol for immunoprecipitation, detecting PPIs often proves challenging. Protein degradation is the primary cause of this issue, which we must carefully avoid. This protocol is simpler and less expensive than other techniques for examining PPIs, and does not include freeze following in the process, thus reducing protein degradation as much as possible.

Approximately 48 hours after transfection of HEK 293 cells, wash the cells thrice with ice cold PBS. Add 500 microliters of protein lysis buffer, containing protease inhibitors into the dish. Collect the cells using a cell scraper and add them to a tube with low protein absorption on ice.

Vortex the tube every five minutes and continue incubation on ice for 10 minutes. Centrifuge the samples at 16, 400 G for 10 minutes at four degrees Celsius. Collect the supernatant and transfer it into a fresh 1.5 milliliter tube with low protein absorption.

After determining the protein concentration, separate 200 to 1, 000 micrograms of protein in a fresh tube. Add protein lysis buffer to adjust the final volume between 500 and 1, 000 microliters. To begin, add 40 microliters of one to one protein G gel slurry into the tube, containing the protein sample.

Rotate the sample for one hour on a rotator in approximately 30 seconds cycles at four degrees Celsius. Next, centrifuge the sample at 12, 000 G for 20 seconds at four degrees Celsius. Place the tube on ice for one minute to allow the beads to level off.

Collect the supernatant and transfer it into a new 1.5 milliliter tube with low protein absorption. Remove the aliquot for input into a separate 1.5 milliliter tube. Add four times the concentrated sample buffer to the input to make an equal dilution, and heat at 98 degrees Celsius for 10 minutes.

Next, add 20 to 30 microliters of one-to-one epitope tag slurry to the sample. Rotate the sample for two hours on a rotator in approximately 30 second cycles at four degrees Celsius. Centrifuge the epitope tagged protein sample at 5, 000 G for 30 seconds at four degrees Celsius.

Wait one minute on ice to allow the beads to level off. Then discard the supernatant. Add one milliliter of protein lysis buffer containing protease inhibitor into the tube, and mix the content.

Rotate the tube on a rotator for approximately 30 second cycles at four degrees Celsius for five minutes. Centrifuge again, and place the tube on ice for one minute to level off the beads before discarding the supernatant. Now, add 10 microliters of sample buffer, and boil the sample at 98 degrees Celsius for 10 minutes.

Centrifuge the sample at 5, 000 G for 30 seconds at four degrees Celsius. Place a column in a new tube with low protein absorption and transfer the gel to a column using a pipette with a cut tip. Centrifuge at 9, 730 G for one minute at four degrees Celsius and collect the flow through.

Place the gel in the electrophoresis chamber and add tris-glycine SDS buffer up to the appropriate volume around the gel. Load the alluded protein samples onto a sodium dodecyl sulfate poly acrylamide gel. Perform electrophoresis at 100 volts and 400 milliamp.

Transfer the gel onto a polyvinylidene fluoride membrane, and block the membrane block with 5%skim milk for one hour at room temperature. Wash the membrane thrice with PBS, polyoxythelene, sorbitan monolaurate on a shaker at top speed for five minutes. Finally, incubate the membrane with the primary antibody overnight on a shaker at four degrees Celsius.

In transfected HEK 293 cells, immuno blotting confirmed the association between DYKDDDDK-PRDM16, and HA-EHMT1 in groups transfected with DYKDDDDK-PRDM16 and HA-EHMT1.