The overall goal of this procedure is to isolate and detect poid related neuronal proteins from cultured neurons using a simple and adaptable biochemical assay. This is accomplished by extracting and immobilizing the target protein in the presence of ethyl or NEM to ensure the blocking of all free thiol groups. The second step is to treat the immobilized target protein with hydroxyl mean or ham, to cleave the thioester linkage between the palmated cysteine residue and the palmitate molecule.
This frees up the Palmated cystine style group and makes it available for labeling in the third step. The immobilized target protein is incubated with biotin B-M-C-C-A file specific molecule tagged with biotin, this biotin elates and tags, the Thile group that was originally pyl. The final step is to detect the level of pation of the target protein by SDS page and western blotting for Strp Aden.
Ultimately, the IP A BE assay is used to show levels of the lipid palmitate reversibly attached to a neuronal target protein, and can also detect subtle changes in target proteins. Pation levels. The main advantage of this technique over existing methods like detection of protein, palm mutilation using metabolic labeling with trait A palmate is that the acyl biotin exchange assay is more sensitive and less time consuming.
The A BE assay also allows for more quantitative estimates of palm mutilation and is optimally suited for detecting small changes in the levels of palm mutilation. Though this method can provide insight into identifying palmated proteins and cultured hippocampal neurons, it can also be applied to other systems such as primary neural cultures from other brain regions, heterologous cell lines, and even primary tissues. To begin extraction of target protein from cultured primary hippocampal neurons, prepare the lysis buffer and NEM solution as described in the written procedure accompanying this video.
Next, combine the lysis buffer and NEM solution by adding 0.5 milliliters of two molar NEM solution to a fresh 50 milliliter conical tube. Add 20 milliliters of lysis buffer over the top. Always add the NEM ethanol solution first and the lysis buffer Second, so as to properly mix the two in a four degrees Celsius cold room, quickly place the mixture on a rocking platform to fully mix for five minutes.
Remove cultured primary rat hippocampal neurons from the incubator instructions on how to culture. The neurons can be found in the text after placing the cultured neurons on ice. Gently remove the cellular media.
Gently wash the neurons twice with cold phosphate buffered saline. Next, add 300 microliters of lysis buffer plus 50 millimolar NEM to the first well of hippocampal neurons and scrape cells using a disposable cell lifter. Collect the cell lysate and then discharge the lysate into the next well of that group.
Scrape the cells within the second well using the cell lifter. Collect and repeat using a third well if needed. To further concentrate the lysate, collect the cell lysate into a pre-cool 1.5 milliliter micro centrifuge tube.
Add 100 microliters of lysis buffer plus 50 millimolar NEM to the first well and repeat the weld scraping process to collect any remaining cell lysate. Pass the total cell lysate through a 26 and one half gauge syringe five to six times to aid in mechanical lysis. Being careful not to blow bubbles into the solution.
Mutate the cell lysate for at least 30 minutes at four degrees Celsius following centrifugation. Collect the cleared cell lysate in a new pre-cool 1.5 milliliter tube on ice. Repeat the demonstrated harvest protocol for all groups of cells.
Measure protein concentration in all lysate samples using the BCA assay. As per manufacturer's instructions. Normalize the volume of all lysate samples from groups of cells to have exactly equal protein with a recommended minimum of 500 micrograms.
Top up all samples with lysis buffer plus 50 millimolar NEM to 500 microliters total volume. Next, add one to five micrograms of primary antibody directed against the target protein to each lysate sample. Mutate the antibody lysate mixed samples overnight at four degrees Celsius before precipitating and immobilizing a target protein.
Prepare a 50%slurry of protein A or protein G coated SRO speeds as described in the written protocol. As a final step, add an equal volume of 50%slurry to each antibody lysate sample, and mutate for at least one hour at four degrees Celsius. In performing ael, exchange the proper pH and temperature of all the buffers and reagents as well as their freshness are critical.
To ensure a successful experiment. Begin the ham cleavage with preparation of a number of tubes with lysis buffer of different phs. The pH is very important for these steps and should always be adjusted.
Using a pH meter per sample, prepare two milliliters of lysis buffer pH 7.2 and 0.5 milliliters of stringent buffer prepared in lysis buffer. Also prepare 0.5 milliliters of lysis buffer plus 10 millimolar NEM per sample. ADD PMSF and protease inhibitor tablets to all lysis buffers.
Hydroxyl mean or ham is used to cleave the thioester linkage between the palmated cysteine and the palmitate fatty acid. This is essential to free up the palmated cysteine style group and make it available for labeling with biotin. The omission of ham cleavage can therefore serve as a useful negative control.
Prepare additional 1.5 milliliter tubes on ice labeled as the minus ham control for each sample following the incubation of the antibody with the cell lysate. And then with the beads, gently centrifuge all sample beads at 0.5 times G for one minute at four degrees Celsius with the tubes on ice. Remove the supernatant and resus suspend the beads in 600 microliters of lysis buffer plus 10 millimolar NEM.
After Resus suspending all of the sample beads immediately collect 200 microliters of the samples lysate beads slurry, and discharge into that sample's minus ham tube on ice, leaving 400 microliters of each sample's lysate bead slurry in the tube for the plus ham sample. This is to account for the limited degradation of the target protein caused by ham treatment. One third of the beads are used for the minus ham treatment and the remaining two thirds are used for the plus ham treatment.
Add an additional 300 microliters of lysis buffer plus 10 millimolar NEM to the minus ham sample. Pipette an additional 100 microliters to the plus ham sample for a total of 500 microliters in each sample. Incubate the tubes for 10 minutes on ice and then proceed to wash the minus ham and plus ham samples as described in the written procedure following sample wash at 0.5 milliliters per sample of lysis buffer pH 7.2 to all minus ham samples.
Add 0.5 milliliters per sample of ham buffer to all plus ham samples. Then mutate all samples for one hour at room temperature. Do not exceed one hour following ham treatment.
Perform acyl biotin exchange by biotin BMCC labeling as discussed in the written procedure. Once biotin BMCC labeling is complete, gently wash the samples as described in the text. Remove the supernatant of the final wash, leaving the pelleted beads in a slurry with remaining buffer in the bottom of the tube.
Dip a pipette with a small diameter tip into the very bottom of the tube through the slurry and quickly collect any remaining buffer. Being careful not to take up any beads. Add 40 to 50 microliters of two x sample buffer plus five millimolar DTT to each sample.
If necessary. Vortex the beads to completely mix with the sample buffer and immediately centrifuge at high speed to collect all beads in a pellet submerged in sample buffer. After boiling the samples and centrifuging again, add the complete volume of eluded protein in the supernatant from each sample to a single lane within a poly acrylamide gel suitable for western blotting.
Using SDS page, organize all samples so that the minus ham control elu and plus ham elu for a single sample are run immediately adjacent to one another. During SDS page following SDS page transferred to A-P-V-D-F or NITROCELLULOSE membrane, begin western blotting with washing and blocking as described in the written procedure, prepare an antibody solution of TBST plus 0.3%BSA, add streptavidin HRP antibody, which has been reconstituted at one milligram per milliliter in distilled water. Wash the membrane once in TBST for 10 minutes.
Then incubate the membrane in STREPTAVIDIN antibody solution on a rocking platform for either one hour at room temperature or overnight at four degrees Celsius. Wash the membrane three times in TBST for 10 minutes on a rocking platform. In order to detect the pation signal, expose the HRP luminescence using a chemiluminescent substrate kit in order to normalize pation levels to the amount of the protein of interest that was immuno precipitated.
Strip the membrane using Western blott stripping buffer for five to 10 minutes on a rocking platform at room temperature. Repeat western blotting steps using the primary antibody against the protein of interest that was originally used for the immunoprecipitation. Quantify pation of the protein of interest using image analysis software and normalize pation levels to the amount of immuno precipitated protein.
The I-P-A-B-E assay specifically detects thile pation of cystine residues along substrate proteins and can be used to detect pation of immuno precipitated neuronal proteins. Treatment of the immuno precipitated neuronal protein with ham cleaves, the thioester linkage between palmitate and Sistine Thal group allowing for specific incorporation of biotin BMCC onto the newly available file group, which can be subsequently detected using western blotting. The omission of ham cleavage prevents the incorporation of biotin BMCC and functions as a negative control for specific poid biotin elation of the plus ham sample.
Therefore, the minus ham control should always be run adjacent to the plus ham sample for western blotting by SDS page. In an optimized experiment, the pation signal of the neuronal protein delta catenin can be readily detected by blotting for biotin BMCC at a concentration of one micromolar using streptavidin conjugated with HRP against parallel minus and plus ham samples. The specific pation signal for delta Kain appears at its predicted size of 160 kilodaltons only in the plus hand sample.
The specificity of this signal was confirmed by re probing for delta CATENIN with the specific antibody that was initially used to immuno precipitated resulting in a signal in both ham treatments at the same predicted size. Optimization of the I-P-A-B-E assay will result in a western blotting profile of a minus ham sample that is readily distinguishable from the plus ham sample and exhibits minimal to no pation signal. The concentration of biotin BMCC used to label PALMATED CYSTINES requires careful optimization and if a super saturating concentration of biotin BMCC is used during the A BE chemistry steps, excessive background and non-specific signals will be visible.
The resulting stripped avid in Western blob profiles among the minus and plus ham samples for delta catine and palm mutilation appear similar when treated with four micromolar biotin BMCC with additional background signals at different sizes, which indicates that inappropriate biotin occurred. Because hydroxy lamine is a powerful reducing agent that results in limited degradation of the target protein, it is necessary to normalize the amount of immuno precipitated protein used for minus and plus ham samples. Normalization requires the use of double the amount of immobilized target protein in the plus versus the minus ham sample, which results in indistinguishable western blotting profiles for the target protein as shown for delta catenin.
However, if the amount of immobilized target protein is not normalized between minus and plus ham samples, the resultant western blot signal for the target protein in the plus ham sample can be lost as shown for delta catenin when equal amounts of protein were used in both ham treatments. After watching this video, you should have a good understanding of how to extract your chosen neuronal protein from cultured hippocampal neurons. Use a BE chemistry to detect if it is a substrate for palm mutilation in minimal time and be able to adapt this protocol for use in other cell lines and tissues.
