Pull-down Assay of Biotin-labeled Histone peptides
Binding Buffer
50 mM Tris pH 7.5
150-300 mM NaCl
flowable0.05% NP-40
1.Thaw GST-tagged proteins on ice. Spin at 13K for 10 min, and carefully take proteins
from the top of the solution. Avoid taking any Glutathione pharo beads with your samples!
2.Dilute proteins for binding: each binding requires 1-2 µg of GST-tagged protein innew
300µl of binding buffer. For 8 binding assays, make 3 ml of binding buffer contains 10-20 ug GST-protein which includes a tube (300ul) of fusion protein without histone peptide as negative control. Also save a tube as input.
3.Add 1 µg different biotinylated histone peptides (1mg/ml) into each tube. (not the
input tubes)history是什么意思
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4.Rotate at 4o C for 4 h to O/N.7一12岁手工科技小制作
资本增值5.Prepare the Streptavidin Sepharo beads (Amersham). U 12-15 µl Streptavidin
beads for each binding assay. Invert the bottle gently to mix the beads well before taking beads. For 30 assays, using a cut tip P1000 to take about 0.4-0.45 ml of the slurry and transfer to a 15 ml Falcon tube, bring the volume to 5 ml -10 ml with cold binding buffer, spin the beads at 500g for 3 min (2K rpm of JA 5.3 rotor for 2 min).
Remove the supernatant and wash the beads at least two more times with binding buffer. After the final wash, bring the volume to 0.9 ml and resuspend the beads.
cet查分6.Add 30 µl of 50% slurry into each tube. Rotate at 4˚C for 1 hr.
7.Spin at 2-4K for 1-2 min at table-top centrifuge. Save the supernatant as Sample FT
(Flowthrough) if needed.三级人力资源管理师
厍8.Wash the beads with 1 ml of Binding Buffer for x times. Rotate at 4oC for 5 min for
each wash.
9.Resuspend beads in 60 µl of 2x SDS sample buffer. Boil it and ready for SDS/PAGE.
archyFor 10% input, take 30 ul of samples from the saved input tube, add 30 ul 2x SDS sample buffer and boil. Load 10 µl for each Western.
