Acyl-resin assisted capture or Acyl-RAC is a sensitive and reliable method that can be used to detect protein S-acylation in a variety of biological samples. This protocol bypasses some of the limitations of metabolic labeling and radio labeling, and allows the simultaneous detection of S-acylation of multiple proteins. Not only live cells but also primary tissues and frozen samples.
S-acylation can regulate a variety of cellular processes such as protein trafficking, plasma membrane targeting, signal transduction, iron transport, and protein-protein interactions. It is important to use freshly prepared hydroxylamine solution with carefully adjusted pH for each experiment to ensure an efficient and specific cleavage of the thioester bond. With a demonstration of this method it's critical for steps like chloroform, methanol precipitation.
The pancake shape pellet obtained during this step should be handle very carefully. It can be fragile and partial loss of the sample during the step can lead to uneven sample recovery. Demonstrating the procedure will be Savannah West, a graduate student from our laboratory.
To obtain cell lysates, collect the cells of interest into a conical centrifuge tube. And remove any cell debris by centrifugation. Wash the pellet in 5 milliliters of PBS.
And immediately resuspend the cells in 600 microliters of freshly prepared lysis buffer. Agitate the sample at 1, 500 revolutions per minute in a thermo shaker for 30 minutes at four degree Celsius. Before clearing the lysates, pellet detergent and soluble material by centrifugation.
At the end of centrifugation, collect the cleared lysate in a pre-cooled 1.5 milliliter microcentrifuge tube on ice. And perform a Bradford or bicinchoninic acid assay to estimate the protein concentration according to standard protocols. Add methanol and chloroform to lysate at a 2:1 ratio.
Shake rigorously to create a homogenous suspension and centrifuge the sample to collect a protein pellet at the interface between the aqueous and organic phases. Tilting the tube, use a needle or a gel loading tip to aspirate as much solvent as possible. Air dry the protein pellet for a few minutes.
Before gently mixing the tube contents with 600 microliters of methanol, taking care not to break up the pellet. After carefully removing the methanol wash, dry the protein pellet on a bench top for approximately five minutes. Next, resuspend the protein pellet in 200 microliters of 2SHB buffer.
And vortex at 42 degree Celsius and 1, 500 revolutions per minute in a thermo shaker. When the pellet is dissolved, add 200 microliters of 0.2%MMTS in 2SHB to the sample. And incubate the protein for 15 minutes at 42 degree Celsius and 1, 500 revolutions per minute in a thermo shaker.
At the end of the incubation, perform 3:4 chloroform-methanol precipitations as demonstrated. To remove the MMTS, dissolve the pellet in 100 microliters of fresh 2SHB buffer with vortexing. And dilute the sample with 300 microliters of buffer A after each precipitation.
After the final precipitation, dissolve the samples in 200 microliters of 2SHB buffer and dilute with 240 microliters of buffer A.Measure the protein concentration again and set aside a 40 microliters from each sample as input controls. Split samples into two equal volumes of 200 microliters. And mark the tubes as plus hydroxylamine and minus hydroxylamine.
Add 50 microliters of freshly prepared neutral two molar hydroxylamine to a final concentration of 400 millimolar to the plus hydroxylamine tube. And 50 microliters of neutral two molar sodium chloride to the negative control, a minus hydroxylamine tube. Then add 30 microliters of TS bead slurry to each tube.
And rotate the tubes for one to two hours at room temperature. At the end of the incubation wash the beads four times with 1%SDS in buffer A to remove any residual hydroxylamine. After the last wash, wash all of the bead samples with three gentle one minute microcentrifugations.
Carefully aspirating the supernatants and resuspending the beads in 500 microliters of 1%SDS in buffer A at each wash. After the last wash, gently spin down the beads as demonstrated. And aspirate as much supernatant as possible without disturbing the beads.
To recover the proteins from the beads, add 50 microliters of 4%SDS sample buffer to each tube and incubate the samples at 80 degree Celsius and 1, 500 revolutions per minute for 15 minutes in a thermo shaker. At the end of the incubation, let the samples cool before centrifuging to completely pellet the beads. Then use a gel loading tip to transfer the eluted proteins into new 1.5 milliliter tubes.
And run the samples on an SDS-PAGE gel to analyze the S-acylation of the proteins of interest by Western blotting. Tyrosine kinase Lck can be detected in lysates treated with neutral two molar hydroxylamine. to cleave the thioester bond between cysteine residues and the fatty acid moiety.
Stripping and reprobing with antibodies against proteins Fyn and LAT demonstrate that acyl-resin assisted capture assay can be used to analyze S-acylation of multiple proteins at the same time. In addition, S-acylation of Lck, Fyn, and LAT can be readily detected in primary mouse splenocytes. Indicating that this modification is conserved between two species.
In addition to the identification of novel S-acylated proteins, this method can also be used to assess changes in protein S-acylation under different biological conditions. The number of newly identified S-acylated proteins has increased tremendously after the double up meant a fast, and reliable technique such as Acyl-RAC. In addition to the identification of novel S-acylated proteins, this method can also be used to assess changes in protein S-acylation under different biological conditions.
