Biochemistry
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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Chapters
Summary June 30th, 2019
We characterized a new kinase protein using robust biochemical approaches: Western Blot analysis with a dedicated specific antibody on different cell lines and tissues, interactions by coimmunoprecipitation experiments, kinase activity detected by Western Blot using a phospho-specific antibody and by γ[32P] ATP labeling.
Transcript
Immunoprecipitation is a very powerful technique to isolate and purify a target protein. In smooth conditions, proteins, regulators, or substrates may be co-immunoprecipitated. A new interaction network may then be discovered.
The activity of the immunoprecipitated protein may also be assessed. In particular, activity of of a kinase may be tested on different substrates by P32-ATP labeling. Nevertheless, activity of inhibitors targeting a kinase involved in a disease may be evaluated.
They may constitute lead compounds to develop new drugs. This method can be extended from mammals to yeast or bacteria. This technique is not that difficult.
To key points:ensure to have a negative control to be sure the detected interaction is specific, and carefully choose lysis conditions to keep interactions and activity. Small details and gestures are important to ensure the success of this technique, and they are never described in materials and methods of articles. Demonstrating the procedure will be Fabienne Godin, a technician from our laboratory.
After cells are prepared in the cell culture room within the biosafety cabinet, use a 10 milliliter pipette to remove the media from the plates and discard it in a dedicated waste bottle with bleach. Then, add 10 milliliters of fresh supplemented DMEM, and put the plates back into an incubator at 37 degrees Celsius. To prepare the transfection mixture, in a 15 milliliter tube, add 450 microliters of 10 millimolar Tris hydrochloride solution at pH 7.5, supplemented with one millimolar EDTA and 50 microliters of 2.5 molar calcium chloride solution.
Mix by inversion. Into the tube, add a volume corresponding to 10 micrograms of plasma DNA, amplified by a DNA midiprep kit, and diluted with the elution buffer of this kit, and whose concentration has been determined. Invert the tube to mix.
Then, with smooth agitation on a vortex mixer, slowly add 500 microliters of BES buffered saline 2X concentrate, drop by drop into the tube. Move it very carefully, not to disturb the complex formation between DNA and calcium phosphate. Incubate for at least 15 minutes, or up to 45 minutes, at room temperature.
Now, transfer the plate to transfect from the incubator to the safety cabinet. Add the prepared DNA complexes drop by drop onto the cells all over the surface of the plate. Incubate for 24 to 72 hours in the incubator at 37 degrees Celsius.
To prevent protein degradation, prepare lysis buffer on ice, taking into consideration four milliliters of lysis buffer for each transfected plate. Take the plate with transfected cells from the incubator. Remove the media and discard it in a dedicated bleach waste bottle.
To wash the cells, add three milliliters of cold PBS on the side of the plate drop by drop, to avoid detaching transfected cells. And gently swirl the plate to spread the buffer all over the surface. Tilt the plate to remove the PBS and discard it into the waste bottle.
Repeat the washing once more. Place the plate on ice. At the end, carefully remove the rest of the PBS.
Next, add 500 microliters of cold lysis buffer to the transfected washed cells. Spread it all over the surface of the plate. Incubate for 10 minutes on ice.
From time to time, again spread the buffer all over the surface of the plate. After that, use a cell spatula to scrape the cells off the surface and collect them in a microcentrifuge tube. Centrifuge for 10 minutes at 10, 000 times G and four degrees Celsius.
Collect the supernatant lysate into a new microcentrifuge tube and collect a 50 microliter aliquot of this fraction to another new microcentrifuge tube. This aliquot will be used to check if transfected proteins are well expressed. Discard the pellet in the trash.
First, gently resuspend the stock solution of agarose beads coupled with the appropriate antibody. Cut the end of a 200 microliter tip to make a one to two millimeter opening. Attach the tip to a micropipette and draw up 40 microliters of the solution, allowing beads to enter the tip.
Pipette the beads up and down several times to saturate the tip to ensure the correct volume of beads and transfer the beads to a microcentrifuge tube. Add 500 microliters of TNET buffer, mix by inversion, and centrifuge for two minutes at 1, 000 times G and four degrees Celsius. Carefully remove the supernatant, and add 500 microliters of TNET buffer.
Incubate on a rotating wheel for at least one hour at four degrees Celsius. Then, centrifuge the tube for two minutes at 1, 000 times G and four degrees Celsius and wash the beads twice with 500 microliters of lysis buffer by inverting and centrifuging. After washing, transfer the previously collected total fraction lysate into the tube and incubate on a rotating wheel at four degrees Celsius for two to four hours.
Now, centrifuge the tube containing the immunoprecipitated beads for two minutes at 1, 000 times G and four degrees Celsius. Wash the beads five times with 500 microliters of lysis buffer by inverting and centrifuging. When washing the beads, take care not to go too close from the beads while removing supernatant.
Otherwise, beads will be aspirated and at the end of the experiment, no more beads will be left. For elution, first centrifuge for two minutes at 1, 000 times G and four degrees Celsius. Use a one milliliter micropipette to carefully remove the supernatant.
Then, with a Hamilton syringe equipped with a cemented needle, remove the last drops of the supernatant to avoid aspiration of the beads. Next, add 40 microliters of 4X Laemmli buffer. Homogenize by gently tapping the tube.
Incubate for five minutes at room temperature. Then, centrifuge for five minutes at 10, 000 times G and room temperature. With a Hamilton syringe, transfer the supernatant eluate into a new microcentrifuge tube.
Store at minus 80 degrees Celsius. HEK-293 cells were transfected with untagged LIMK 2-1 or one of the HA tagged isoforms of LIMK 2. LIMK 2-1 is well expressed in the different conditions.
Anti LIMK 2-1 antibody does not recognize LIMK 2a and LIMK 2b isoforms, and it is specific, as its signal decreases when cells are transfected with LIMK2 small interfering RNA, compared to control small interfering RNA. In various human cell lines, LIMK 2-1 appeared to be expressed in HEK-293 and HeLa, but not in C6.Coimmunoprecipitation experiments were used to assess the interaction of LIMK2-1 with upstream kinase ROCK. Each of the different proteins encoded by the transfected vectors were well expressed.
The three isoforms of LIMK2, as well as larp6, were efficiently immunoprecipitated. In the eluates, ROCK was detected and thus coimmunoprecipitated with the three isoforms of LIMK2, but not with larp6. The detected interaction is then specific.
Coimmunoprecipitated LIMK 2-1 was tested for its kinase activity via gamma P32 ATP labeling. LIMK 2-1 did not phosphorylate cofilin, whereas it did phosphorylate myelin basic protein, or MBP. LIMK 2-1 is efficiently immunoprecipitated in this test.
It is critical to have negative control while performing immunoprecipitation to be sure the interaction is specific. The composition of a lysis buffer must be carefully set up to preserve interaction and activity. Upon immunoprecipitation, kinase activity may be assessed to count different substrates.
Mutation may be easily introduced and may unravel the role of specific residues. Functional studies may also be performed to understand the physiological role of new highlighted interactions. Cells must be cultured in a dedicated culture room within a biosafety cabinet.
P32 ATP has to be handled with specific caution. Shield to protect from radiation, Geiger counter, specific waste collect, personal breast and finger badges to detect radioactive exposure and filter tips.
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