The overall goal of the following experiment is to extract protein from human and mouse brain tissue for proteomics analysis. This is achieved by using a common lysis buffer to obtain the same protein extraction adapted for two dimensional electrophoresis approaches. Next two dimensional fluorescence difference gel electrophoresis is used to separate proteins for mass spectrometry identification.
This is followed by a miniature 2D gel electrophoresis with immuno blotting to identify post-translational modifications. The results can show global protein expression. Host translational changes all ligamentization and catalytic products based on the differences in isoelectric point and molecular weight.
The combination of 2D dash and 2D monolo can help us to answer key question in the neuro proteomics field by simultaneously providing information on expression changes. Postal modification and catabolism in products Begin with harvesting and homogenizing the brain tissue. Prepare the tissue at 10%weight to volume in extraction buffer for human brain tissue.
Homogenize the tissue using a glass Pipetter, however, use a Teflon homogenizer for rodent tissue to disintegrate either homogenate. Sonicate it at 60 hertz using 30 half second pulses. Next, determine the protein concentration with a Bradford assay using BSA as a standard.
Now gather the solutions for chloroform methanol precipitation in an ice bucket. Plan to collect about 125 micrograms for 11 centimeter IPG strips or about 1.25 milligrams for 18 centimeter IPG strips. Once the solutions are at four degrees Celsius, perform the precipitation completely on ice.
First, add three volumes of methanol, then one of chloroform. Shake this mixture. Next, add three volumes of cold water.
Vortex, this mixture for one minute centrifuge the sample tube at 12, 000 Gs for 30 minutes at four degrees Celsius. Aspirate and discard the supernatant. Then add three volumes of methanol vortex and spin down the proteins in the same manner.
Dry the collected protein pellet under nitrogen gas before 2D DIGE. Re suspend the protein in 2D buffer at 2.5 milligrams per milliliter. Then sonicate the samples as done before until they are used.
Store them at minus 80 degrees Celsius before beginning this step and after protein precipitation. It is necessary to determine the protein concentration once more by Bradford Assay. Following the same procedure as shown earlier before psy dye labeling.
The quality of the protein sample is verified by SDS page for that purpose. Dilute 15 micrograms of protein in LDS and he it to 37 degrees Celsius in a heat bath or block. Then run them on a poly acrylamide gel stain the gel with kumasi blue solution for at least an hour.
Then detain the gel overnight in 7%acetic acid, 10%ethanol solution. Once the protein sample quality is established, the next day the SD labeling procedure is started. First measure the pH of the lysate, which should be basic to neutral.
The optimal pH is 8.6 for subsequent reactions. Next, set up the dye conjugation reactions for each protein sample. Mix 50 micrograms with 400 picomoles of S3 dye and 50 micrograms with 400 picomoles of S five dye.
Do this in quadruplex for a total of eight D coupling reactions per sample. For each tested sample, make a complimentary set of DI coupling reactions using protein from the control such as from a diseased brain. Then for every pair of reactions, set up an internal control reaction with 400 picomoles of CY two and an even representation of all proteins totaling 250 micrograms.
SAI labeling is achieved by incubating all the reaction mixtures at four degrees Celsius in the dark for an hour. After the incubation, combine all the sets of three different cyan reactions into 150 microliter volumes to each set of reactions at 200 microliters of 2D buffer. Then proceed with the 2D electrophoresis steps.
Begin with preparing unlabeled protein solutions for the preparative gel from which protein spots will be excised for each sample and for the positive control. Aliquot 300 to 350 micrograms of unlabeled protein into 2D buffer and allow them to rehydrate in contact with the IPG strip. Next load the IPG strips transfer the labeled and the unlabeled protein to a rehydration.
Well then cover each well with an IPG reaction strip. Follow this with an overlay of mineral oil and allow the strips to passive passively rehydrate with the protein solution overnight at no more than 25 degrees Celsius. The next day, carry out isoelectric focusing of the IPG strips.
Then remove the oil and store the IPG strips at minus 20 degrees Celsius to equilibrate the IPG strips. Submerge them in equilibration buffer for 15 minutes. Then transfer the strips to 4.7%IDO acetamide in equilibration buffer, lacking DTT while bathing the strips.
Use a large 2D gel system to pour four 12%SDS page gels per sample in low fluorescence glass plates for the labeled proteins. Also pour one 12%SDS page gel in 1.5 millimeter thick standard glass plates for each of the unlabeled proteins. These are the preparative gels.
When the gels are cooled, transfer the strips to the top of the gels and overlay the strips with 0.5%Aros once cooled, run the gels at 2.5 watts at four degrees Celsius overnight and analyze them the next day. Many 2D reactions can be carried out with protein samples used for the preparative gels. Measure out less than 100 micrograms of sample into 200 microliters of 2D buffer.
Then vortex the sample mixtures vigorously and give them a quick spin. Load the samples onto 11 centimeter IPG strips and allow them to passively rehydrate overnight covered in mineral oil the following day. Perform the isoelectric focusing.
Then move the strips through three baths of equilibration buffer for 15 minutes per bath. The strips are then run on 2D SDS page gels. Layer each strip onto a precast gel of the appropriate molecular weight.
Poly acrylamide for the protein of interest. Later, transfer the gels to NITROCELLULOSE PVDF membrane and analyze them with antibodies in development of this protocol. Three different lysis.
Buffers were tested. Firstly, a common biochemical and molecular biology buffer. Tris SDS was tested.
Tris SDS had poor resolution compared to another tested buffer UTS. The third buffer to de was ruled out as testable samples could not be prepared with it. Next, human and mouse brain proteomes were investigated.
Human control cortex samples and ad cortex samples were compared. Mouse brain proteins from mice with AD like tau pathology were also viewed from the human sample. SCI two was looked at independently as was S SI three and sci five for each D.The high spot resolution made alignment easy.
Qualitative mini 2D analysis was used to investigate post-translational modifications and a ligamentization alpha synuclein in ad tissue was found to have a normal profile, but also found as a dimer marked by an asterisk. Arrows marked where alpha synuclein was found. Ubiquitinated amyloid precursor protein was also viewed by mini 2D.
Sporadic ad was compared with familial ad. In familial ad beta amyloid one to 42 and ISO variance are lower in concentration because liga forms were found at eight kilodaltons. After watching this video, you should have a good understanding of how to study the proteome in order to compare complex samples by measuring protein expression modifications.
