The overall goal of this procedure is to identify in vivo protein, protein interactions of membrane proteins through a membrane strep protein interaction experiment or membrane spine. This is accomplished by in vivo crosslinking using the reversible crosslinker for formaldehyde. The second step is to purify the strept tagged membrane bait protein together with the cross-linked prey proteins.
Next, the cross-link is reversed by boiling. The final step is the separation of the membrane bait protein and the co purified prey proteins by sodium do desal sulfate, poly acrylamide gel electrophoresis or SDS page. Ultimately, immuno blotting and mass spectrometry analysis is used to identify the prey proteins, allowing analysis of membrane protein interactions.
I'm Sabina Hunka and we will show you today the membrane spine technology. The main advantage of this technology over existing methods like co IP, is that it allows you even to detect low affinity or transient protein protein interactions. Kline Shaana will demonstrate you the procedure.
She's one of my PhD student who developed the Protocol To Begin this procedure, Grow bacterial cells expressing the membrane bait protein with a C terminal strep tag fusion induce the expression of membrane bait proteins at early log phase by addition of 0.5 millimolar IPTG to 500 milliliters of medium to allow sufficient expression. Incubate the bacteria until the late log phase. Prepare to perform formaldehyde cross-linking under a safety fume hood due to the toxicity of the formaldehyde.
Transfer the culture vessel under a safety fume hood. Split each sample into two samples, one omitting cross-linking, and one, including the formaldehyde cross-linking. Add four milliliters of 37%formaldehyde solution to a 250 milliliter culture to reach a final concentration of 0.6%Transfer the culture vessels back and grow the cells for 20 minutes more as before.
Then fill the cultures into centrifuge tubes under a safety fume hood. Collect the cells by centrifugation at 3000 times G for 30 minutes. Remove the supernatant by firstly decanting and secondly by suction.
Using a pipette under a safety fume hood, discard the supernatants disposing of the toxic formaldehyde containing supernatant properly. In order to reach optimal yield during membrane protein preparation, first, prepare Sphero plats to begin gently dissolve two milligrams of lysozyme in 0.1 molar E-D-T-A-P-H eight in a 1.5 milliliter tube. To prepare buffer P two, which should be made fresh on the day of the experiment.
Resus suspend the cell pellets in 10 milliliters of freshly prepared tris sucrose buffer with protease inhibitor, and transfer the solution to a 15 milliliter conical tube. Add one milliliter of buffer P two and incubate on ice for 30 minutes. Collect the Sphero plats by centrifugation at 3000 times G for 30 minutes and discard the supernatant.
Carefully incubate the Sphero plast pellets overnight at negative 20 degrees Celsius working on ice. Resus suspend the Sphero Plast palate in six milliliters of freshly prepared buffer. P three.
Sonicate the sample four times for one minute continuously on ice with a one minute pause between each burst. In order to disrupt the Sphero plast centrifuge the sample at 10, 000 times G for 10 minutes to harvest cell debris following centrifugation. Transfer the supernatant using a pipette to an ultracentrifuge tube.
Pellet the membrane fraction at 100, 000 times G for 30 minutes. Carefully wash the pellet in 20 millimolar tris buffer pH eight without dissolving it. Then drive a tube with a paper tissue avoiding disturbing the pellet at any time.
To resuspend the pellet containing the membranes, add one milliliter of tris, buffer a micro magnetic rod and resuspend the pellet, partly with a pipette stir on ice for one hour, use a 20 microliter aliquot to determine the protein Concentration. Normalize the protein Concentration of the membrane fraction to five milligrams per milliliter with tris buffer pipette 2.5 milliliters of the membrane fraction to an ultracentrifuge tube and add 0.25 milliliters of 20%Triton X 100 to reach a final concentration of 2%in order to solubilize the membrane proteins. Then add a micro magnetic rod and stir on ice for one hour.
Equilibrate a one milliliter strept actin super flow gravity flow column with eight milliliters of buffer. W.Remove the micro magnetic rod from the solubilization sample and ultracentrifuge at 100, 000 times G for 30 minutes to pellet the insoluble membrane fraction after the run is complete, remove the sate using a pipette. For further purification, apply the supernatant to the column using gravity flow.
Wash the column with four milliliters of buffer W Repeating this washing step five times. Elute the membrane bait proteins with one milliliter of buffer E.Repeat this elution. Step four times, concentrate the alluded fractions.
Two, three, and four to 300 microliters with a centrifugal filter unit. Mix 200 microliters of each sample with 50 microliters of five x as DS page loading dye. Split each preparation into 125 microliter aliquots before boiling.
One aliquot of each preparation for 20 minutes at 95 degrees Celsius to reverse formaldehyde cross links. Let the samples cool down to room temperature for at least 10 minutes on the bench. Top load 30 microliters from each sample to a single lane of a poly acrylamide gel suitable for immuno blotting.
Use a prestained molecular weight marker For the best orientation and perform as DS page following transfer of proteins To a nitrocellulose membrane. Block the membrane to prevent non-specific labeling for one hour. At room temperature, dilute and incubate the prey protein specific primary antibody.
Use an HRP linked antibody as the secondary antibody and develop the immuno blot using a chemiluminescent detection kit with high sensitivity. Monitor the signal using a classical film processing procedure or digital imaging equipment. If no specific antibody is available for the prey, protein, or unknown interaction, partners should be identified.
Use mass spectrometry or MS for identification. Silver stain the SDS page using an MS compatible staining kit. According to the manufacturer's protocol, perform all staining and washing steps in glass tanks.
Excise the respective bands before analyzing them by high resolution LCMS as bait Protein. The integral membrane protein CPXA of VISIA coli was used. CPXA is a sensor kinase and consists of an N terminal sensor domain with two transmembrane domains integrating a large extra cytoplasmic sensor domain and a C terminal highly conserved cytoplasmic catalytic domain.
After stimulation, CPXA activates its cognate response regulator, CPXR activated CPXR diffuses off to mediate the response for membrane spine. The strep tag was fused to the C terminus of CPX. A membrane spine analysis reveals that CPXA strep cross-linked to other proteins or protein complexes indicated by a smear in the unveiled formaldehyde treated sample.
Without formaldehyde treatment, this smear is absent. Moreover, a direct protein protein interaction of CPXA strep with its cognate response regulator protein CPXR as the prey protein is only detectable in the presence of formaldehyde. Supporting the specificity of formaldehyde cross-linking with silver staining.
A band which is specific for the boiled sample is apparent. The arrow marks a band that was analyzed by MS.Analysis and which confirmed CPXR as the interaction partner Of CPXA. After watching this video, He should have a good understanding of how to combine pro and cross linking affinity purification at mass spectrometry to identify interaction partners of membrane Proteins.
