The overall goal of this procedure is to detect detergent-sensitive interactions between membrane proteins or other proteins. This procedure requires one protein to be histidine-tagged and over expressed in the cells, while the other is a myc-tagged peptide. This method can help answer question in the field of biochemistry important protein interaction studies.
The main advantage of this technique is the ability to detect the interaction between membrane proteins in a detergent-sensitive way. The procedure is fast and easy. Though this method can provide insight into membrane protein interactions, it can also be used to study weak protein interactions.
We first had the idea of this method when we wanted to check the interaction between the membrane proteins, Saltilin and GLUT4, but attempts to co-immunoprecipitate them from the mammalian lysis remain unsuccessful. To place an order for the peptide, select the target sequence of the peptide critical for interaction and add a myc epitope at the end or C terminus of the sequence. Once the peptide has arrived, pepare 100 micrograms per milliliter of working solution of the peptide in ultra pure water.
Seed three 3T3-L1 pre adipocyte cells in four 10 centimeter culture dishes and keep in the incubator. Next, transfect two culture dishes with the target protein tagged with 6X histodyn and label as HISP in the other two dishes with two control plasmids and label as WT.After transfection, leave the cells in the incubator for 48 hours to reach 80 to 90%confluency. To prepare the cell lysate, wash the cells on ice three times with 10 milliliters of 1X buffered saline cooled four degrees Celsius.
Then, add 500 microliters of lysis buffer to each of them. Then, detach the cells from the dishes using a cell scraper to prepare the lysate. Transfer the cell lysate prepared from each culture dish to four separate 1.5 millimeter tubes and label all the tubes.
Then, pass the cell lysate through a syringe with 26 gauge needle up and down for five times for total cell lysis. Then, centrifuge the cellular lysates at 16000xg for 10 minutes at four degrees Celsius. Transfer the supernatants to four identically labeled new tubes.
For future troubleshooting and control experiments, aliquot 20 microliters of the cell lysate and store it 20 degrees Celsius. For later use, titer the wash buffer at PH8 with hydrochloric acid to obtain PH6 and store the buffer at four degrees Celsius. Next, to prepare a homogenous suspension of the cobalt beads, carefully swirl the bottle.
Then, transfer 40 microliters of cobalt bead suspension to all the individually marked four tubes. After adding the cobalt beads, add one milliliter of lysis buffer to the tubes. Then, centrifuge the tubes at 1000xg for five seconds.
Discard the supernatant and pipette 40 microliters of lysis buffer to the settled beads. Then, transfer the cell lysate to the corresponding tubes and incubate at four degrees Celsius for 90 minutes on a tube rotator. After 90 minutes, centrifuge the samples at 1000xg for five seconds.
Then, collect the supernatant labeled Flowthrough-1 and store at 20 degrees Celsius. Next, add 500 microliters of the wash buffer with PH8 to the individual tubes and re-suspend the beads gently. Centrifuge the tubes at 1000xg for five seconds and expel the supernatants.
Then, add wash buffer with PH8 with or six to the tubes, according to the labels and re-suspend the beads well. Centrifuge the tubes at 1000xg for five seconds and expel the supernatants. Prepare one microgram per milliliter working solution of the peptide and water buffer with PH6 or eight.
Then add 100 microliters of the peptide solution to the washed beads corresponding to similar PH.Then, incubate the beads at four degrees Celsius on a tube rotator for 30 minutes. Then, take four microcolumns, label them and place the columns in collection tubes labeled Flowthrough-2. After the incubation is over, add the incubation mixture to the columns.
Let the solution pass through by gravitational force and collect the sample from the flow. Place the columns in new collection tubes and store at 20 degrees Celsius. Then, pass 500 microliters of wash buffer with either PH6 or eight by gravitational flow through the individual columns and discard the flowthrough.
Repeat this step four times. Centrifuge the tubes at 1000xg for five seconds. Place the column in the new collection tube labeled Elution.
It is important to wash the beads in all the tubes very carefully and equally to get rid of the unbound peptide. To the washed beads, add 40 microliters of tricine sample buffer with beta mercaptoethanol. Let it sit for 20 minutes at room temperature.
Centrifuge the column at 8000xg for two minutes. Discard the columns and heat the collecting tubes at 100 degrees Celsius for 10 minutes. And then, centrifuge it at 1000xg for five seconds.
Alternatively, add 40 microliters of Elution Imidazole buffer to the washed beads and incubate at room temperate for 20 minutes. After the incubation is over, centrifuge the tubes at 8000xg for two minutes. Discard the columns and add 40 microliter tricine sample.
Heat the collecting tubes at 100 degrees Celsius for 10 minutes, and then centrifuge it at 1000xg for five seconds. Use the commercially available 10-20%tricine gradient gels. Use Tris/Tricine/SDS running buffer, then load 20 microliters each of the eluted samples and total lysates on the gel and start running the electrophoresis unit.
Once the electrophoresis is over, use transfer buffer supplemented with 20%methanol to transfer the material from the gel onto 0.45 micrometer nitrocellulose membrane. After blocking the membrane, cut it horizontally. Next, probe the upper portion of the membrane with Anti His P antibody in the lower half of the membrane with anti myc.
To study the interaction between immobilized Sortilin on cobalt beads in GLUT4, immunoblot analysis was performed. Cellular lysates and eluates were obtained wild type and Sortilin-myc/His-tagged transfected 3T3-L1 cells and then loaded on SDS page. The blot was probed with anti-myc antibody.
The blot shows a 110 kilodotin Sortili-myc/His-tagged and five kilotin myc-first luminal loop-GLUT4 from the transfected eluates. Next, to study the importance of PH and the interaction between Sortilin immobilized on cobalt beads and GLUT4, immunoblot analysis was performed. In this assay, Myc-first luminal loop GLUT4 was incubated with beads immobilized with Sortilin-myc/His-tagged proteins both at PH6 and eight.
The blot shows a clear interaction between Sortilin-myc/His and myc-first luminal loop GLUT4 both at PH6 and eight from the eluates obtained from the Sortilin-myc/His-tagged transfected 3T3-L1 cells. Once mastered, this technique can be done in three to four hours until the samples are ready for Western blood analysis. While attempting this procedure, it's important to remember to design peptide that is soluble, preferrably in water.
To strengthen your results, other methods like immunoprecipitation following crosslinking can be performed. After watching this video, you should have a good understanding of how to detect membrane protein, protein interactions in a detergent sensitive procedure. It also allows to explore interaction between protein fragments.
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