Biotinylated peptide pull down assay

Dan Levy's Lab 2018
Lee Elisha

Pull-down assay of biotin-labeled peptides.

Adapted from the Gozani lab, Stanford University.  

Binding buffer: PBSX1

0.1% NP-40

1. Dilute peptides with binding buffer to get a concentration of 0.25ug/ul.

For example: Take 2.5 µl of 4mg/ml peptide stock and dilute into 40ul binding Buffer (0.25ug/ul)

2. Take 5 µl of the diluted peptide (1.25ug) into 200-300μl of binding buffer

3. Add 1 μg of His/GST- fusion proteins; * also prepare negative control with recombinant protein without peptide.

4. Prepare input sample (for WB) Take 10ul out of each assay tube and add PSB. Alternatively- take 5-10% of the recombinant protein and add PSB.

5. Rotate at 4oC for 4 h to O/N.

6. Prepare Streptavidin Sepharose beads.

7. Use 20 μl Streptavidin beads for each binding assay. Wash the beads three times with 1ml binding buffer. Take out sup and add binding buffer so each sample will get 50ul. Rotate at 4oC for 1 h

8. Centrifuge at 3000rpm 2min and discard FT.

9. Wash beads three times with 1ml binding buffer

10. Take out sup and add 40ul SBX5. Boil at 95 oC 5min. Take 10 ul for western blot and followed by incubation with anti-His/GST antibodies.