IP and immunoblot analysis

Protocols

IP and immunoblot analysis

IP of cells in 6-well plates as an example:

1. Prepare the lysis buffer I (20mM Tris–HCl, pH8.0, 0.5%Nonidet P-40, 10mM NaCl, 1mM EDTA) containing 1mM Na₃VO₄, 1mM PMSF and complete protease inhibitor cocktail (catalog no. 04693132001, Roche, USA). Add 300ml of the lysis buffer to each well (of note: Add Na₃VO_4, PMSF and cocktail before lyzing the cells). 
2. Scrap cells with lysis buffer I and collect into 1.5ml tubes. Cell lysates were lyzed on ice for 20min, followed with centrifugation at 14000 rpm at 4°C for 15 min.
3. Transfer supernatants to new tubes; aspirate 48ml of the supernatants as input. Add 20ml protein A/G agarose (catalog no. sc-2003, Santa Cruz, USA) together with primary antibodies (anti-HA or anti-Flag for 1mg and anti-cGAS was diluated at 1:100) into tubes, followed by incubation at 4°C overnight. 
4. On day 2, wash precipitates at least 5 times with cell lysis buffer I by centrifugation at 6000rpm at 4°C for 1 min. At last, all the supernatants of precipitates were aspirated with syringe needle, the protein precipitates were added with SDS loading buffer and boiled at 95°C for 10min. 
5. After heating denaturation, analyze protein samples by 10% SDS-PAGE.

Procedures for immunoblot analysis

1. Prepare 10% SDS-PAGE containing resolving and stacking gel detail as follows:

2. Load protein samples to 10% SDS-PAGE gel. 

3. Start electrophoresis with constant voltage (resolving gel at 90v and stacking gel at 120v). Terminate the electrophoresis once the blue running line reached to the bottom of the gel.

4. Activate the PVDF membrane with methanol for 15s, followed with equilibration with transfer buffer for at least 2min (put on gloves and avoid touching it with your finger).

5. Equilibrate gel in transfer buffer on shaker. 

6. Assemble the “sandwich structure” in order with sponge, filter paper, gel, PVDF, filter paper and sponge. Make sure [-] electrode (black) / gel / PVDF / [+] electrode (red or white). 

7. Start the transfer with constant voltage at 100v for 90min on ice. 

8. After the transfer, block PVDF membrane with 5% nonfat milk in 1×PBS-T for 1h at room temperature (RT).

9. After the block, wash the PVDF and incubate with primary antibody in 1% BSA, overnight at 4°C.

10. After the incubation, wash the PVDF membrane with PBS-T for 10min×3 times on shaker. 

11. Incubate with secondary antibody in 1% BSA at RT for 1h. 

12. After the incubation, wash the PVDF with PBS-T for 10min×3 times on shaker.

13. Incubate PVDF transiently with ECL, detect and capture signals by imaging instrument.

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