GST-protein binding assays

GST pull-down C.elegans extract

Preparation of GST fusion proteins:

  1. Transformation in BL-21
  2. IPTG induction (2 hrs at 37 °C) in 50 mL culture
  3. Keep pellet at -20 °C
  4. Resuspend frozen pellet in 5 mL STE buffer + 5mM DTT + 1.5 % sarcosyl + protease inhibitors and incubate on ice 30 min.
  5. Sonicate 3X 45 sec. on ice. Add Triton X-100 1% and incubate on ice 30 min.
  6. Centrifuge at 15 550 g, 25 min at 4 °C.
  7. Aliquot soluble fractions and flash freeze at -80 °C.

Quantification of GST fusion proteins:

  1. Add 20 uL of glutathione beads to 500 uL fraction for each fusion protein and incubate O/N at 4°C or 1 h at RT.
  2. Wash 3X in STE buffer.
  3. Add 20 uL of SDS-PAGE loading buffer and load 10 uL on gel with BSA (0.25-1 ug) for Coomassie staining.

Pre-binding of GST fusion proteins to beads:

  1. Binding of (1-5 ug) of GST fusion proteins to 20 uL of glutathione sepharose beads in STE buffer, O/N at 4 °C.
  2. Next day, wash beads 3X with lysis buffer C (25 mM HEPES-KOH, pH7.5, 150 mM NaCl, 10% glycerol, 1 mM MgCl2, 1 mM DTT, 2 mM EDTA, 0.5% Triton X-100 and protease inhibitors) and incubate in this buffer on ice.

Preparation of C.elegans extract:

  1. Homogenize worm pellet (300-500 uL) in 3 volumes of lysis buffer C with the dounce homogenizer (30-40 shots).
  2. Centrifuge lysate at 17 000g, 10 min at 4 °C.
  3. Transfer supernatant to a clean tube and repeat step 2.
  4. Pre-clear lysate with glutathione beads, 1h at 4 °C and measure protein concentration with Bradford assay.
  5. Add 2 mg of worm lysate to prebound GST fusions proteins.
  6. Incubate 4 hrs at 4 °C. Add 10 ug of RNase A after 2 hrs.
  7. Wash the beads 6X with 3X bed volume of lysis buffer C.
  8. Elute in 20 uL of SDS loading buffer by heating 5 min at 65 °C.

Load on gel and blot for potential interactors.

 

Source: Thivierge et al. NSMB 2011