The overall goal of this procedure is to test proteins that bind to Cal Modlin in a calcium dependent manner. This is accomplished by first collecting proteins of interest. Next calmodulin sero speeds are prepared for protein binding.
Then the proteins are incubated with calmodulin sero speeds. Finally, the proteins that bind to in a calcium dependent manner are alluded. Ultimately, results can be obtained that show proteins that bind to calmodulin in either the presence or absence of calcium through western blood analysis.
The main advantage of this technique over other existing methods, such as calm chromatography and immuno precipitation, is that it requires less protein and less time. This method can help answer key questions in the cow modlin signaling field, such as what proteins bind to cow modlin in a calcium dependent manner, and how post-translational modifications can affect their binding to cow modlin. Visual demonstration of this method is critical as a bead handling steps are essential and the description of the resus and retention of the beads is difficult to follow without seeing it.
To prepare tissue for homogenous inject organotypic hippocampal slices with a virus containing a plasmid expressing the recombinant protein of interest. In this example, the recombinant protein is GFP tagged Neuro Grandin. Allow the tissue to express the protein overnight approximately 12 to 18 hours after viral injection, add one milliliter of dissection buffer to a Petri dish.
Transfer the cultured tissue with insert to the Petri dish and add two milliliters of dissection buffer to the insert to submerge the tissue using a scalpel. Collect five to 10 organotypic hippocampal tissue slices by gently scraping the tissue free from the insert membrane With an inverted pasture pipette. Transfer the suspended tissue to a 1.5 milliliter micro centrifuge tube.
Next, centrifuge the samples at 1500 RCF for one minute. To separate the tissue from the dissection buffer, carefully remove the supernatant by aspiration. Make sure not to disturb the pellet.
For each slice used, add the tissue to 30 to 60 microliters of homogenization buffer with a one milliliter pipette with a cut tip, and then homogenize it thoroughly using a pestle before returning the homogenate to its original tube. In order to remove cellular debris, centrifuge the remaining homogenate at 1100 RCF for 10 minutes and carefully collect the snat by aspiration while avoiding contamination from the pellet. Aliquot 10%of the supernatant as a sample of the input.
Store the remaining supernatant on ice during preparation of the cal sero beads for use. Later, for each pull down pipette 400 microliters of suspended cal Modlin Sero speeds into a two milliliter micro centrifuge tube with a relatively flat bottom to maximize surface area and interaction of solutions with the beads during the incubations, rotate the micro centrifuge tube on its side to wet the beads, and then centrifuge the beads at 21, 000. RCF for 30 seconds.
And carefully remove the supernatant by aspiration. Make sure not to disturb the beads to wash the beads at 100 microliters of the respective homogenization buffer containing either two millimolar calcium chloride to beads being used to pull down calcium cal modlin binding proteins. Or two millimolar E-D-T-A-A known calcium chelator to beads being used to pull down apo cal modlin binding proteins.
Gently tap the tube to Resus. Suspend the beads and centrifuge at 1500 RCF for one minute. Carefully remove the supernatant by aspiration, making sure not to disturb the beads to bind proteins to calmodulin spheros for pull down.
Begin by splitting the SNAT into two equal volumes. Depending on the condition per aliquot, add the appropriate amount of calcium chloride or EDTA to the supernat to a final concentration of two millimolar. Add the supernatant to the washed beads in the appropriate homogenization buffer.
Gently tap the tube to mix. Incubate the samples at four degrees Celsius for three hours on a shaker. We suspend the beads every 30 minutes or so to increase the efficiency of binding.
Next centrifuge the samples at 1500 RC F for three minutes. Remove 50 microliters of supernatant as a sample of unbound protein and carefully remove the remaining snat by aspiration and discard. Wash the beads three times as before with 100 microliters of the appropriate homogenization buffer.
The most difficult step in this procedure is the elucian of target proteins in an efficient manner. The best way to ensure success is to use the right kind of tube to minimize bead loss and to appropriately resuspend the beads in elution buffer warming. The elution buffer beforehand can also help significantly To elute the protein at 50 microliters of elucian buffer to the beads.
Samples that were homogenized and bound in buffer containing calcium are alluded in buffer containing EDTA and samples that had buffer containing EDTA are alluded with calcium. Incubate the beads at room temperature for 30 minutes on a shaker. Mix the beads by gently tapping the tube about every five minutes.
Centrifuge the beads at 1500 RCF for three minutes and carefully remove the 50 microliters of SNAT for the bound protein sample by aspiration. Make sure not to disturb the beads. Carry out SDS page and western blotting to identify the protein of interest as a positive control in the opposite condition.
Prop Pro protein known to bind Cal Modlin. This figure shows an example of a Cal Modlin pull down assay testing Cal MODLIN binding of GFP TAG Neuro Grandin compared to endogenous neuro Grandin. The homogenous input shows that GFP neuro Grandin was expressed in addition to endogenous neuro grandin and calcium calmodulin dependent kinase two as expected based on the known binding of endogenous neuro Grandin.
GFP tagged Neuro Grandin was alluded in the absence of calcium and did not bind in the presence of calcium. In contrast, the control calcium calmodulin dependent kinase two was alluded only in the presence of calcium and was unbound in its absence. This shows that the calmodulin beads were functioning properly and the EEU were efficient.
Most importantly, this shows that GFP neuro grin binds to Apoch Cal Modlin in a similar fashion to the endogenous form, suggesting that the GFP tag did not alter the function of a recombinant protein. Once mastered this technique, not including SDS page and Western blood analysis can be finished in six hours when done properly While attempting this procedure, it's important to remember to label all samples and tubes for the appropriate condition because any change in calcium concentration can compromise the entire experiment. After watching this video, you should have a good understanding of how to efficiently determine the ability of particular proteins to bind a CAL module in a calcium dependent manner.
By collecting the proteins of interest, incubating them with the appropriately prepared co modulant SRO speeds, eluding the bound proteins and testing them using Western blood analysis.
