Pulldown assay

Protocol:

1. Transfection of 293T cells

1. Replate HEK 293T cells onto 6 well-plates (I like to do 4 wells for each condition) at a confluence of around 60%.
2. The next day, cells should be at around 80-90% confluence.
3. Transfect cells with the following transfection master mix per well (incubate master mix 15 minutes after mixing components together before adding to cells):
   1. 250 uL serum free, antibiotic free DMEM
   2. 7.5 uL TransIT 293
   3. 2.5 ug of plasmid
4. Collect cell lysates 24 hours later using lysis buffer and use immediately for co-IP
   1. For 6-well plates, I typically aspirate the cell medium/transfection mix prior to adding 500 mL of lysis buffer to each well

2. Co-IP of Txnip Using agarose beads

a. 24 hours prior to collecting cell lysates, bind antibody of interest (anti-V5) to Protein A/G agarose beads, following the manual

1. I use around 10 ug of antibody for each 1 mg of agarose beads
2. Weight out the apprproiate amount of b for co-IP
3. Wash beads with 1 mL of C1 and vortex.
4. Place beads on magnetic rack and remove supernatant.
5. Add the appropriate volume of antibody + lysis buffer and vortex.
   1). (lysis buffer and Ab should be 100 uL per beads)
6. Incubate on a roller at 37°C overnight
7. Place beads on magnetic rack and remove the supernatant.
8. Wash beads with PBS (800 uL if less than 20 mg) 3 times.
   1). Place beads on magnetic rack and remove supernatant with each wash.
9. Wash beads with PBS for 15 minutes at room temperature on the roller.
10. Place beads on magnetic rack and remove the supernatant.
11. Resuspend beads in 100 uL PBS per mg beads for a final concentration of 10 mg beads/mL.

b. Transfer antibody-coupled beads to a new tube (1.5 mg per reaction).

1. Wash the beads in 900 uL of lysis buffer.
2. Place beads on magnetic rack and remove the supernatant.
3. Resuspend the antibody-coupled beads in cell lysate (I use 1.5 mg of cell lysate per 1.5 mg of beads).
4. Incubate on a roller at 4°C for 10-30 minutes.
5. Place beads on magnetic rack and remove the supernatant.
6. Wash the beads in 200 uL of lysis buffer three times (DO NOT VORTEX).
7. Wash the Dynabeads in 200 uL lysis buffer and incubate on the roller for 5 minutes at room temperature.
8. Transfer the bead suspension to a new tube.
9. Place beads on magnetic rack and remove the supernatant.
10. Resuspend the beads in 60 uL PBS and incubate on a roller for 5 minutes at room temperature.
11. Transfer the supernatant to a clean tube and use for gel electrophoresis.

3. Western Blot of co-IP samples

1. Take 20 uL of co-IP sample and transfer to a new tube.
2. Add 8 uL of NuPage LDS buffer and 4 uL 2-mercaptoethanol to each sample.
3. Boil at 95°C for 5 minutes and then transfer samples to ice.
4. Assemble the gel electrophoresis cassette and fill with 1X MES Running Buffer.
   1. Load the samples onto the gel with 8 uL of PageRuler Plus Prestained Protein Ladder.
5. Run the gel at 150-200V for 30 minutes-1 hour on ice.
6. Transfer the protein to a PVDF membrane using the semi-dry transfer using the following setup (top to bottom): 
   1. Transfer pad
   2. Gel
   3. PVDF membrane (activated with 100% MeOH)
   4. Transfer pad
7. Run the semidry transfer at 25V for 35 minutes.
8. Stain the gel with coomassie blue stain and dry after washing with water.
9. Wash membrane with 0.1% PBST and block with 5% milk for 1 hour.
10. Incubate membrane in primary antibody at the appropriate dilution in 5% milk for 1 hour at room temperature or overnight at 4°C if signal is not that strong.
11. Wash membrane in 0.1% PBST for 5 minutes 4 times to remove residual antibody.
12. Incubate membrane in the secondary antibody at the appropriate dilution in 5% milk for 1 hour at room temperature.
13. Wash membrane in 0.1% PBST for 5 minutes 4 times to remove residual antibody.
14. Develop membrane using ECL reagents.
15. Expose and develop films in the dark room.

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