Sample preparation for FLAG pull down

Sample preparation for FLAG pull down

This protocol describes the procedures for preparing samples for FLAG pull down to identify FLAG-tagged target protein interactors.
Note: For transcription factors, their transcriptional activity typically involves binding to genomic DNA. However, it’s hard for traditional IP buffers to fully dissolve chromatin. Here, we employ MNase to digest chromatin, which allows us to capture more chromatin-associated proteins.

Materials

Reagents

• MNase (Thermo Fisher #EN0181)
• Benzonase (Sigma #70664-3)
• Anti-FLAG M2 Magnetic Beads (Sigma #M8823)
• RNase A (Thermo Fisher, #12091021)

Buffer

• Buffer A (10 mM HEPES pH 7.5, 10 mM KCl, 1 mM MgCl2, 0.1% Triton X-100, 1 mM DTT, Protease Inhibitor)
• IP Lysis Buffer (20 mM Tris-HCl pH 7.5, 140 mM NaCl,  1% NP-40, 2 mM EDTA, Protease Inhibitor)
• Wash Buffer 1 (10 mM HEPES pH 7.5, 1 mM DTT)
• Wash Buffer 2 (50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.1% Triton X-100)
• MNase Digestion Buffer (10 mM HEPES pH 7.5, 10 mM CaCl2, 1 mM MgCl2)
• Benzonase Digestion Buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 100 mM MgCl2)

Day 1

1.Block Anti-FLAG M2 Magnetic Beads with 1% BSA in PBS at 4℃ for 3 hours.
2.Harvest 293T cells expressing the target protein and place the cells on ice.
3.Add IP Lysis buffer (3 PCV, packed cell volume ), and incubate for 30 minutes at 4℃.
4.Centrifuge at 12,000 rpm, 4℃ for 20 minutes. Transfer the supernatant (cytoplasmic fraction) to a new tube.
5.Wash the pellet once with Wash Buffer 1, centrifuge at 12,000 rpm, 4℃ for 5 minutes, discard the supernatant, and grind the pellet (chromatin fraction) using a glass rod.
6.Add 500 µL MNase Digestion Buffer and 0.3 µL MNase. Digest chromatin at 37℃ in a water bath for 1 hour, mixing gently every 15 minutes. 
7.After digestion, add NaCl to a final concentration of 150 mM, NP40 to a final concentration of 0.1%, EDTA to a final concentration of 2 mM, and EGTA to a final concentration of 2 mM. Incubate on ice for 15 minutes.
8.Centrifuge at 12,000 rpm, 4℃ for 15 minutes.
9.Mix the supernatant with the cytoplasmic fraction from step 4, then centrifuge again.
10.Place the blocked beads on a magnetic stand, discard the supernatant, resuspend the beads with the protein sample, add 2 µL of RNase A, and incubate overnight at 4℃ with rotation.

Day 2

1.After the overnight incubation, place the beads on a magnetic stand and discard the supernatant.
2.Add 250 µL cold Benzonase digestion buffer and 0.1 µL Benzonase. Incubate at 4℃ with rotation for 30 minutes.
3.Wash beads four times with wash buffer 2. The sample can be used for further mass spectrometry sample preparation.