产生和人类INO80染色质重塑复合物及其亚纯化

The aim of this procedure is to generate and purify wild type and mutant versions of the multi subunit in oh 80 Chromatin remodeling complex. This is accomplished by first isolating the nuclei from cells expressing flag epitope tagged in O 80 proteins In the second step, nuclear extracts are prepared, and then the wild type or mutant human in O 80 chromatin remodeling complexes are purified by Anti-Flag aros chromatography. Ultimately, the subunit compositions of the purified NO 80 complexes can be analyzed by western blot or silver staining.

This method can help answer key questions about how the subunits of MultiPro chromatin remodeling complexes contribute to their overall activity. This method’s designed specifically for studying wild type and mutant versions of the NO 80 Chromatin Remodeling complex, but it can also be used to study other multi subunit nuclear complexes. Like the mediator.

The most critical step of the procedure is the nuclear extraction step. People usually runs into problem because during the salt extraction step, people tend to add salt too quickly or too vigorously After culture In roller bottles, transfer the HEC 2 9 3 cells to a suitably sized graduated conical tube and spin them down for 10 minutes at 1000 times G and four degrees Celsius. After removing the supinate, measure the volume of the packed cell pellet, then re suspend the pellet in five pack cell volumes of buffer A with gentle pipetting and incubate the cells on ice for exactly 10 minutes.

After spinning down the cells again, measure the cell pellet a second time, it should have increased up to twofold in size. This time, re suspend the cells in two pack cell volumes of buffer A then transfer the cell suspension to an appropriately sized down tissue homogenizer and homogenize the cell suspension with the loose glass pestle until 90%of the cells stain positively with trian blue centrifuge, the homogenized cells in a 45 milliliter high speed centrifuge tube for 20 minutes at 25, 000 times G and four degrees Celsius in a fixed angle rotor. Then remove the SUP natant containing the cytosolic proteins from the nuclear pellet to extract the nuclei with salt.

Now add 2.5 milliliters of buffer C, but every three milliliters of starting packed cell volume to the pellet. Then using a serological pipette, dislodge the pellet from the wall of the tube and transfer the entire mixture to a down homogenizer of appropriate size. Homogenize the mixture with two strokes of a loose pestle to resus suspend the nuclei and then transfer the resuspended nu nuclear fraction into a chilled beaker of such a size that the suspension will fill the beaker to at least 0.5 centimeters deep.

Next, calculate the volume of five molar sodium chloride needed to bring the solution to a final concentration of 0.42 molar sodium chloride according to the following formula. Now gradually add the calculated volume salt solution drop by drop while gently starring the suspension with a glass rod on ice to increase the salt concentration of the suspension until all of the sodium chloride has been added and the solution becomes very viscous. Then carefully transfer the viscous suspension into 70 milliliter polycarbonate tubes in a Type 45 TI rotor with cap assembly and use a mutator to slowly rock the sealed tubes at four degrees Celsius After 30 minutes, ultracentrifuge the samples in a TI 45 rotor for 30 minutes at 40, 000 times G and four degrees Celsius, and pull the nuclear extract containing supernatants into a single plastic container.

Then divide the nuclear extract solution into conveniently sized aliquots snap, freeze them in liquid nitrogen and store them at minus 80 degrees Celsius. To prepare Anti-Flag aros for immuno purification, begin by using a 200 microliter pipette with the tip cutoff to transfer 200 microliters over a 50%Anti-Flag aros bead slurry into a 1.5 milliliter micro centrifuge tube. Transfer the pre-washed beads into a conical tube containing 10 milliliters of freshly thawed nuclear extract before hours of four degrees Celsius with slow rotation on a laboratory rotator, and then collect the flag aros by centrifugation for five minutes of 1000 times G and four degrees Celsius.

Next, wash the bead extract P in 10 milliliters of lice, four 50 buffer for five minutes of four degrees Celsius with gentle rocking on a mutator. Then after spinning down the bead extract mixture, once again reuse. Suspend the pellet in 100 to 150 microliters of fresh lice four 50, transfer the beads to a 1.5 milliliter micro centrifuge tube.

Continuing to rinse the 15 milliliter conical tube with 100 to 150 microliter quats of Lysol 50 until all the beads have been transferred to the micro centrifuge tube. After spinning down the beads in a micro centrifuge, wash them three times with one milliliter of lys four 50 and once with one milliliter of EB 100.Buffer. Then to elute the bound proteins, add 200 microliters of EB 100 buffer containing flag peptide and incubate the sample at four degrees Celsius on a nutt.

After half an hour, pellet the beads in the micro centrifuge you gain and then transfer the SUP natant containing the eluted in OAC C complex to a fresh micro centrifuge tube. After repeating the elucian four more times, pass the pulled eluate through an empty spin column to remove any residual flag aero beads from the protein fraction. Finally, use a centrifugal ultra filtration device with a 50, 000 molecular weight cutoff to concentrate the alluded protein fraction approximately 10 fold.

As illustrated in this figure, the purity of the NO 80 complexes prepared using the indicated flag tag subunits, as just demonstrated, can be assessed using silver stained SDS poly acrylamide gels. Any proteins that appear in this control lane are contaminants that bind non-specifically to flag aeros. These labels indicate the bans corresponding to the NO 80 subunits tested, and these indicate the mobilities of the molecular weight markers.

The position of the wild type NO 80 is indicated by the black square, and the positions of the NO 80 mutants are indicated by the open circles. The lanes separated by the black line are from separate gels. The composition of the complexes can be assessed by Western blott using antibodies against various NO 80 subunits.

For example. Here are the subunits of the end terminal domain module, the Helicase center associated module and the SNF two module were assessed. Once mastered, this technique can be finished within one day, beginning with the harvesting of cells and finishing with the purification of complexes.

This technique will allow researchers to purify multi subunit complexes and sub complexes for mammalian cells in order to study their functions. After watching this video, you’ll have a better understanding at how to purify chromatin remodeling and other multi subunit protein complexes from human cell or other momentum cell lines.