Western blotting and immunoprecipitation

Western blotting (WB) protocol:

1.      Confluent cultured cells with or without indicated treatments (e.g. 1x10^6 differentiated THP-1 cells per well in 12-well plate treated with LPS for 3 hrs) washed with ice cold PBS once.

2.      Add RIPA lysis buffer with protease inhibitor cocktail, PMSF and sodium orthovanadate directly on the culture plate. Use 100 ul RIPA lysis for 2x10^6 cells, pipette and transfer to 1.5 tube.

3.      Centrifuge at 12000 RPM, 4 C, for 15 min.

4.      Transfer supernatant into another tube, and quantify protein concentration using DC protein assay.

5.      Add sample loading buffer, and heat the sample for 3-5 min at 95 C, then put on ice.

6.      Load around 30 ug of total lysate in 5-20% SDS-PAGE gel, and SDS-page running buffer. For Nacalai gels, we used 220 V, 30 mA for 50-60 min run (gel running depends on the gel company)

7.      Transfer the gel to 0.45 uM PVDF membrane (Amersham, GE healthcare) using Semi-dry transfer system (Bio-Rad).

8.      Block the membrane using 5% skim milk (some targets like p-IRF3, block using 3% BSA) at room temperature (RT) for 1.5 hrs.

9.      Wash the membrane with TBS-T 3 x 5 min

10.  Add primary antibody in suitable diluent. We mainly used can get signal solution 1 from Toyobo. However, for certain targets as p-IRF3 and Arid5a, we used 3% BSA.

11.  Incubate the primary antibody with gentle shaking (40 round/min) for overnight.

12.  Wash the membrane with TBS-T 3 x 5 min

13.  Add secondary antibody in suitable diluent. We mainly used can get signal solution 2 from Toyobo. However, for certain targets as p-IRF3 and Arid5a, we used 5% skim milk.

14.  Wash the membrane with TBS-T 3 x 5 min

15.  Add the Electrochemiluminescence (ECL) reagents according to the company recommendations, read the membrane using suitable Chemiluminescence machine. First, start with Auto, then you can adjust the exposure time manually if you need.

Immunoprecipitation (IP) protocol:

1.      Confluent cultured cells in 10 cm dish with or without indicated treatments washed with ice cold PBS once.

2.      Add RIPA lysis buffer with protease inhibitor cocktail, PMSF and sodium orthovanadate directly on the culture plate. Use 100 ul RIPA lysis for each 10 cm dish. Scrape the cells and transfer to 1.5 ml tube.

·       Pro tip: at this stage usually, the lysate is viscous and has lot of DNA, which may interfere with the downstream applications. Sonication is important to shear the DNA and make the lysate much clearer. We sonicate with cycling pattern; 3 sec sonication at the high level followed by 30 sec rest for 20 cycles. Sonication shall be done in ice-water not to heat the sample.

3.      Centrifuge at 12000 RPM, 4 C, for 15 min.

4.      Transfer supernatant into another tube.

5.      Add 25-50 ul of the same beads you will use for Immunoprecipitation (IP) to the lysate for pre-clear. Rotate the samples at medium speed at 4 C for 30 min to 1 hr.

6.      Remove the beads by using centrifugation for agarose beads or magnet for magnetic beads. Transfer the lysate to a new tube and add the primary antibody at the desired concentration. Rotate the samples at medium speed at 4 C for overnight.

·       Pro tip: use low binding or siliconized tubes for maximum protein recovery, especially in small reaction volumes.  

·       Pro tip: adding the antibody overnight enhance the recovery of the protein of interest

·       Pro tip: using small reaction volume (e.g. 100-150 ul) can save your reagents of beads and antibodies.

7.      Add 25-50 ul of agarose or magnetic beads to your reaction.

8.      Rotate the samples at medium speed at 4 C for 30 min to 2 hr.

·       Pro tip: don’t keep the beads more than 2 hrs otherwise the background maybe high.

9.      Recover the beads containing antibody by using centrifugation for agarose beads or magnet for magnetic beads.

10.   Wash the beads with the same lysis buffer (e.g. RIPA) 3 times.

·       Pro tip: use rotor for washing the beads. It enhances the wash process and decrease the background.

16.  Elute the protein from the beads using 1x sample buffer diluted in the same lysis buffer. Heat the sample for 3-5 min at 95 C, then put on ice.

·       Pro tip: For elution, use thermomixer (e.g. Eppendorf thermomixer C). This enhances the elution step and the recovery of your proteins from the beads

11.  Continue for WB starting from point 7 in the WB section.

·       Pro tip: For secondary antibody incubation for IP with rabbit-produced primary antibody, use Anti-rabbit IgG (Conformation Specific). This eliminates the light and heavy chain bands of the primary antibody, and it is a must if your target is located within similar molecular weight as the IgG light and heavy chains. 

For the detailed catalog numbers and solutions for WB and IP, please refer to the “Western blot and Immunoprecipitation” of the materials and methods section. For the details of the primary antibodies used in our study, please refer to “Table. S2. List of Antibodies used for western blotting and immunoprecipitation.”  Metwally et al., Sci. Signal. 13, eaay0574 (2020).

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