The gradient fixation method, in which a glycerol gradient centrifugation is performed in the presence of a cross-linker, helps to identify interactions between proteins that bind transiently to multi-subunit complexes. The use of fixation reagents in the glycerol gradient stabilize the bind of loose factors, without the formation of precipitates, allowing the identification of the interactions of specific proteins with spliced semi complex's. The demonstration of end protocol helps first time users to learn information that may be difficult to understand from written text alone.
For yeast total extract preparation, grow the yeast cells expressing one of the splicing factors fused to the tap tag and one liter of Y and B glucose medium supplemented with the appropriate amino acids or nucleic bases. When the culture reaches an optical density at 600 nanometers of one, collect the yeast cells by centrifugation in three 500 milliliter centrifuge bottles, and wash the cells two times in 10 milliliters of cold sterile water per wash. After the second wash, resuspend the cells in one 10th of the cell volume of cold buffer A, and freeze 50 microliter drops of the yeast cell suspension in liquid nitrogen.
After freezing, grind the frozen cell pellets through six cycles at 20 hertz for three minutes in a ball mill device, immersing the container of frozen cells in liquid nitrogen at the end of each cycle. After the last cycle, place the tubes containing the extracts into water at room temperature with occasional shaking. Once melted, centrifuge to collect the extracts.
And quantify the protein that the cleared supernatants by the BCA method. Then fast freeze aliquots of the extracts in liquid nitrogen for minus 80 degrees Celsius storage. To prepare a glycerol gradient, add a 0.1%final concentration of the cross-linking agent glutaraldehyde to a 30%glycerol and buffer A solution, and mixed to homogenize.
Add six milliliters of cold 10%glycerol and buffer A solution to a 12 milliliter centrifuge tube and use a gradient master device syringe equipped with a long needle to layer the 30%glycerol glutaraldehyde solution at the bottom of the tube. Use a gradient master device to create a linear glycerol gradient according to the manufacturer's specifications. Carefully layer 200 microliters of a 7%glycerol and buffer A cushion to the top of the gradient before adding approximately two milligrams of cell extract to the top of the tube.
After glycerol gradient generation, place the sample in a pre cooled swing bucket rotor and collect the extract by centrifugation. Aliquot the sample from each 12 milliliter sample tube in 24 500 microliter fractions, and use a peristaltic pump to push a 40%glycerol solution through the 10 to 30%gradient from the tube to the fraction collection. Then load 50 microliters of each fraction directly onto the nitrocellulose membranes on a dot blot, followed by addition of blocking buffer, and use a primary antibody against CBP to detect the protein on the immuno blot, and an appropriate secondary antibody as necessary.
In the absence of cross-linker CWC 24 is concentrated between fractions five and 13, corresponding to complexes of about 80 to 200 megadaltons. In the presence of the cross-linker, however, the position of CWC 24 on the gradient is shifted to fractions at the bottom of the gradient corresponding to larger complexes. Comparing CWC 24 sedimentation to that of the U5 snRNP subunit, Prp8, and the NTC subunit, Prp19 reveals that CWC 24 is concentrated in the same fractions.
But, because it associates transiently with the spliceosome, it is also present in the lighter fractions. Interestingly, fractions 11 and 12 are those in which Prp8 and 19 start to concentrate, suggesting that this is the portion of the gradient where the B Act complex begins to sediment. Western blot analysis of fractions 11 and 12 reveals Prp8 and 19 signals as smears that barely enter an 8%acrylamide gel, showing that they are indeed part of large complexes.
CWC 24 is also present in fraction 12, but in a much lower concentration, consistent with its transient binding to the B Act complex. Taken together, these results show that glutaraldehyde can be used as a cross-linker to stabilize the binding of transient factors to splicing sub complexes. This method can be applied to the study of any dynamic motor sub-unit complexes that associates transiently with some of their components, such as the spliceosome.
