CoIP BUFFERS AND SOLUTIONS

Transcription

1 CoIP BUFFERS AND SOLUTIONS IP BUFFER [RIPA Buffer (50 mm NaCl) + 25% Acetonitrile] In 500 ml of dh2o add- 250 ml Acetonitrile 50 ml 1M Tris-HCl ph 8.0 (50 mm) 10 ml 5M NaCl (50 mm) 10 ml NP-40 Substitute (1%) 5 g sodium deoxycholate (0.5 %) 1 g SDS (0.1%) Adjust volume to 1L with dh2o SDS-PAGE RUNNING BUFFER (10X, 1L): In 800 ml of dh2o add g Tris Base g Glycine 10.0 g SDS Adjust volume to 1L with dh2o TRANSFER BUFFER (1X, 1L) In 800 ml of dh2o add- 200 ml methanol 3.0 g Tris Base 14.4 g Glycine TBS-T (10X, 1L) In 800 ml of dh2o add g Tris Base 80.0 g NaCl 2.0 g KCl Adjust ph to 7.4 Add 5 ml of Tween-20 Adjust volume to 1L with dh2o

2 COIMMUNOPRECIPITATIONS (v5.4) Note that all volumes/amounts described in this protocol are for a single CoIP. Scale the volumes/amounts accordingly for each particular experiment. DAY 1 STEP 1: HARVEST AND WASH CELLS o Transfer the cells from the culture dish to a 15 ml conical tube o Centrifuge the tube at 500g for 2 minutes o Remove the supernatant o Wash the cells by resuspending the pellet in 5 ml of PBS o Centrifuge the tube at 500g for 2 minutes o Remove the supernatant from the cell pellet o Gently resuspend the cell pellet with 1.5 ml of PBS o Transfer the resuspended cells to a microcentrifuge tube o Centrifuge the tube at 500g for 2 minutes o Remove the supernatant, leaving the cell pellet as dry as possible STEP 2: CYTOPLASMIC PROTEIN EXTRACTION o NOTE: THE FOLLOWING VOLUMES ARE APPROPRIATE FOR A CELL PELLET THAT IS APPROXIMATELY ul o Add 1000 ul of ice cold CER-I to a pre-chilled microcentrifuge tube o Add 10 ul of 100X Halt Protease Inhibitor Cocktail to the CER-I, vortex to mix o Transfer the CER-I/Protease Inhibitor solution to the cell pellet o Vortex the tube vigorously for 15 seconds to resuspend the pellet o Incubate the tube on ice for 10 minutes o During the incubation, use the FAST COOL setting on the refrigerated centrifuge to cool it to 4 C o Add 55 ul of ice cold CER-II to the tube o Vortex the tube vigorously for 5 seconds o Incubate the tube on ice for 1 minute o Vortex the tube vigorously for 5 seconds o Centrifuge the tube at 16,000g for 5 minutes at 4 C o Transfer the supernatant (cytoplasmic fraction) to a pre-chilled microcentrifuge tube, store on ice for later use (or -80 C for long-term storage)

3 STEP 3: NUCLEAR PROTEIN EXTRACTION o Add 500 ul of ice cold NER to a pre-chilled microcentrifuge tube o Add 5 ul of 100X Halt Protease Inhibitor Cocktail to the NER, vortex to mix o Transfer the NER/Protease Inhibitor solution to the nuclei pellet o Vortex the tube vigorously for 15 seconds o Incubate the tube on ice for 40 minutes, vortexing vigorously for 15 seconds every 10 minutes o Centrifuge the tube at 16,000g for 10 minutes at 4 C o Immediately transfer the supernatant (nuclear fraction) to a pre-chilled microcentrifuge tube, store on ice for later use (or -80 C for long-term storage) STEP 4: PRECLEAR BEAD INCUBATION AND SAMPLE SEPARATION o Add 25 ul of Protein-G agarose beads to the nuclear fraction o Incubate tubes at 4 C for 30 minutes with gentle rocking o Centrifuge tubes at 3000g for 3 minutes at 4 C o During the centrifugation, add 750 ul of IP Buffer to each of two tubes o Add 7.5 ul of 100X Halt Protease Inhibitor Cocktail to each tube of RIPA Buffer, vortex to mix o After the centrifugation is complete, transfer the precleared supernatant equally into the two tubes of RIPA Buffer (250 ul of supernatant into each tube) STEP 5: IP - PRIMARY ANTIBODY INCUBATION o Add 5 ul of the primary antibody to one tube (IP) o Add 5 ul of generic IgG to the second tube (mock IP) o Incubate at 4 C overnight with gentle rocking

4 DAY 2 STEP 1: REMOVE THE CELL LYSATE SAMPLE o Remove 50 ul of the IP solution and transfer to a pre-chilled microcentrifuge tube and store on ice. This is the cell lysate sample o Add 50 ul of 2X SDS-PAGE loading buffer to the cell lysate sample o Add 5.2 ul of β-mercaptoethanol to the cell lysate sample o Add 10 ul of glycerol to the cell lysate sample o Vortex the tube briefly o Centrifuge the tubes briefly o Boil the sample for 5 minutes o Vortex the tube briefly o Centrifuge the tube briefly STEP 2: IP - PROTEIN-G AGAROSE BEAD INCUBATION o Add 50 ul of resuspended Protein-G agarose beads to each IP tube o Incubate for 3 hours at 4 C with gentle rocking STEP 3: WASH BEADS AND PREPARE FOR SDS-PAGE o Add a Protease Inhibitor Tablet to 10 ml of IP Buffer and store on ice o Centrifuge the tubes in the Galaxy centrifuge for 10 seconds o Remove the supernatant o Resuspend each bead pellet in 1 ml of IP buffer o Centrifuge the tubes in the Galaxy centrifuge for 10 seconds o Remove the supernatant o Repeat the wash and centrifugation four additional times o After the final wash, completely remove the supernatant o Add 50 ul of IP buffer to the pellet o Add 50 ul of 2X SDS-PAGE loading buffer to each pellet o Add 5.2 ul of B-mercaptoethanol to each pellet o Add 10 ul of glycerol to each pellet o Vortex the tubes briefly o Centrifuge the tubes briefly o Boil the samples for 5 minutes o Vortex the solution briefly o Centrifuge the solution briefly

5 STEP 4: SDS-PAGE AND TRANSFER o Load protein ladder (~2.5 ul per well) and samples (~30 ul per well) into the gel o Run the gel at 200V for minutes (watch the bottom dye band) o Presoak the nitrocellulose paper in Transfer Buffer o After the gel has run, assemble the transfer cassette (from bottom to top: black side of transfer cassette, sponge pad, filter paper, gel, nitrocellulose paper, filter paper, sponge pad, clear side of transfer cassette) o Place the assembled transfer cassette into the transfer tank. Clear side of the transfer cassette towards the red side of the transfer tank. o Add a stir bar and the ice pack into the transfer tank. Fill with Transfer Buffer o Transfer the proteins at 100V for minutes with stirring STEP 5: BLOCK THE BLOT o Before the transfer is complete, make the Block Solution by dissolving 5 g of non-fat dry milk in 100 ml of TBST (5% solution) o After the transfer is complete, disassemble the transfer cassette and remove the nitrocellulose sheet (blot) o Cut the sheet into the appropriate strips and remove excess paper o Briefly rinse the blots with Milli-Q water o Place the blots in a plastic tub and cover with Block Solution (approximately ml) o Incubate at 4 C for 2-3 hours with gentle agitation STEP 6: PRIMARY ANTIBODY INCUBATION o For each blot, pipette 5 ml of Block Solution and 5 ml of TBST into a petri dish o Add 5 ul of the primary antibody (1:2000 dilution) to the solution in the petri dish o Place the blocked blot into the petri dish, protein side down o Incubate overnight at 4 C with gentle agitation

6 DAY 3 STEP 1: WASH OFF THE PRIMARY INCUBATION o Remove the blots from the petri dish, place in a plastic tub and cover with TBST (approximately 50 ml) o Incubate the blots at room temperature for 15 minutes with gentle agitation o Pour off the wash and replace with fresh TBST, repeat the above wash steps 3 additional times o ALTERNATIVELY, you can perform six 5-minute washes if you are short on time STEP 2: SECONDARY ANTIBODY INCUBATION o For each blot, pipette 10 ml of Block Solution into a petri dish o Add 1 ul of the secondary antibody (1:10,000 dilution) to the solution in the petri dish o Place the washed blot into the petri dish, protein side down o Incubate at room temperature for 2 hours with gentle agitation STEP 3: WASH OFF THE SECONDARY ANTIBODY o Wash the secondary antibody from the blot following the exact same procedure as Step 1 above STEP 4: CHEMILUMINESCENCE AND DETECTION o A few minutes before the last wash is complete, prepare the chemiluminescence reagent by mixing equal volumes of the two solutions together and vortexing. Each full blot (2 sides) requires 3 ml total of chemiluminescence solution o Place the blots protein side up on a sheet of Parafilm o Overlay the blots with the chemiluminescence solution o Incubate at room temperature for 5 minutes o Remove the blots from the Parafilm and place them into a plastic sheet protector o Use the BioRad ChemiDoc system to detect the protein bands