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Retesting Y2H interactions by GST pulldown

Transfection of 293T cells

  • Split the cells into 6 well plates 24 h before transfection to get 50-70% confluence one day after (use 3 ml of 10% FCS DMEM/well). For example, one 10 cm dish has the surface area of one 6-well plate. A confluent 10 cm dish can be split into 3 6-well plates.
  • Follow the protocol of the transfection reagent, and transfect the following for each protein pair:
    • A-GST + B-Myc
    • empty-GST + B-Myc
    • B-GST + A-Myc
    • empty-GST + A-Myc

Cell lysis

  • Aspirate medium.
  • Wash the cells off with PBS.
  • Pellet the cells, and resuspend in 300 µl of Lysis Buffer.
  • Keep on ice for 15 min.
  • Spin down at 13000 rpm for 10 min.
  • Take supernatant.

GST pulldown

  • To 120 µl of lysate, add 20 µl of glutathione beads (Amersham) + 500 µl lysis buffer.
  • Incubate 1 hour at 4°C.
  • Wash the beads 3 times with 500 µl of lysis buffer.
  • Dry the beads with thin tips.
  • Add 15 µl of Laemli buffer onto the beads.
  • Boil for 3 minutes before loading the gels.

Gel loading

For each protein pair, run 2 gels as follows:

  • Gel 1, to be probed with anti-Myc, containing the pulldowns of the 4 transfections.
  • Gel 2, to be probed with anti-GST and anti-Myc consecutively, containing straight lysates of the 4 transfections.

Western blotting

Use 0.75% TBST for all steps.

  • Block for 1 hour at room temp in 5% milk.
  • Incubate with primary antibody O/N at 4°C.
  • Wash 3x quick, then 3x 5 minutes.
  • Incubate with secondary antibody for 30-60 minutes at room temp.
  • Wash 3x quick, then 3x 5 minutes.
  • Develop and expose.

Quenching the peroxidase activity between GST and Myc westerns

Rather than stripping the straight lysate blot after GST detection, it is easier to quench the peroxidase activity of the secondary antibody with Azide. This is possible because the two primary antibodies are from different organisms.

  • Incubate the blot for 30-60 minutes in 10 mM NaAzide in PBST
  • Rinse in PBST
  • Perform second primary incubation.

Antibodies

  • Mouse anti-Myc (9E10 Sigma M-4439) diluted 1:2000
  • Rabbit anti-GST (Sigma G-7781) diluted 1:2000
  • Sheep-anti-Mouse (Amersham) diluted 1:5000
  • Donkey-anti-Rabbit (Amersham) diluted 1:5000

Lysis buffer

0.5%
2 0 mM
150 - 200 mM
1 mM
1 tablet / 40 ml

NP40
Tris-Cl, pH 8.0
NaCl
EDTA
protease inhibitors