Good thing takes time

Step 1: Preconjugation of ATM antibody
antibody( Novus: NB100-104) 2.5 ug
Protein A - sepharose slury 30 uL
TBST 200 uL
in 0.5mL tube

o/n 4 degree.

Step 2: reaction( depend on your purpose)

Step 3: ATM IP by Protein A-ATM antibody complex bead
reaction (about 100-50 uL) + complex
4 degree 2 hours.
wash.

Step4: Kinase reaction

ATM IP complex + P53-GST fusion peptide(10 ug)
buffer C: 10uL
150 mM KCL: 10 uL
GammaP(32) ATP: 1 uL

32 degree for 30 min.

Step 5: GST poul-down

Glutathione bead: 30 uL
4 degree, 2 hours

Step 6: 60 uL SDS lameli buffer boiling

Tricks: using long tip to suck out all the buffer in the beads.

wash by TBST 1-2 times


A Luminol/iodophenol chemiluminescent detection system for western immunoblots