小鼠 TrueBlot® Set (with IP beads)

Mouse IgG TrueBlot® ULTRA is provided as 1000X solution. In order to conserve reagent, we recommend incubating the blots from minigels in sealed bags (removing as much air as possible) with minimal volume (2-3 mL). If used conservatively at 2.5 mL/blot, cat. 18-8817-31 (50µL) will yield enough reagent for 20 blots. Note that there are three key procedural considerations: 1. Protein A or G beads may be used with the mouse, goat and sheep TrueBlot secondaries but not with the rabbit TrueBlot secondary. Use of protein A or G beads with the rabbit TrueBlot will result in contaminating bands. 2. Immunoprecipitate should be completely reduced. 3. Milk should be used as the blocking protein for the immunoblot. Components. 1. Mouse IgG TrueBlot®. An HRP-conjugated second step reagent reacting with goat IgGs for optimal signal detection in immunoprecipitation/immunoblotting experiments 2. Anti-Mouse Ig IP Beads: 2.5 ml. Binds 1 mg Ig/ml beads 3. Western blot incubation tray Special Notes: Upon initial use of the IP beads, we recommend that the vial be inverted several times to get the beads into suspension. We recommend to use a large bore pipet to pipet up the liquid for use. For storage of the opened vial of beads, we recommend that the vial cap be sealed with parafilm to help prevent evaporation of the buffer.

Preparation of Cell Lysate
1. Harvest approximately 1x10^7 cells by using cell scraper and transfer to conical tube. If working with adherent cells you can skip this step and lyse directly on the plate (see Step 6)
Note: The total number of cells per mL and the cell equivalent loaded per lane of gel should be optimized specifically for each protein and antibody. Alternatively, protein concentration can be determined using Bradford/Lowry or other protein assay.
2. Wash cells with 10 mL of cold PBS and centrifuge at 400 xg for 10 minutes at 4°C.
3. Discard the supernatant and repeat step 2.
4. After the second wash, remove the supernatant and resuspend the cell pellet in 1 mL of cold Lysis Buffer containing protease Inhibitors (such as a cocktail- see recipe below). Final concentration of cells should be about 1x10^7 cells/ml.
Note: If using adherent cells, the cold Lysis Buffer can be added directly to the plate and put on a rocker at 4°C. Harvest by either scraping or collecting just the supernatant and proceed to Step 8.
5. Gently vortex/mix and transfer to 1.5 mL tube.
6. Place on ice for 30 minutes, with occasional mixing.
7. Centrifuge at 10,000 xg for 15 minutes at 4°C.
8. Carefully collect the supernatant, without disturbing the pellet and transfer to a new clean tube and discard pellet.
9. The protein concentration can be determined by Bradford or other assay. Samples can be diluted to 1 µg/ µL.
10. The cell lysate can be frozen at this point for long-term storage at -80°C.

Cell Lysate Preclearing
1. Resuspend the immobilized Protein A or Anti-Goat IgG bead slurry by gently vortexing. Remove 50 µL and wash in Lysis buffer or IP buffer, if different. Resupend in 50 µL IP buffer.
2. Add 500 µL of cell lysate (5x10^6 cells or 500µg protein) to the pre-equlibrated bead slurry to and incubate on a rocking platform or a rotator for 30-60 minutes at 4°C.
3. Centrifuge at 2,500 xg for 2-3 minutes at 4°C and transfer the supernatant to a new 1.5 mL tube. If any of the bead slurry has been transferred, centrifuge again and carefully transfer the supernatant to another fresh 1.5 mL tube.

Procedure Step I: Immunoprecipitation
1. Add 1-10 µg of immunoprecipitation antibody to the tube containing the cold precleared cell lysate.
Note: This concentration of monoclonal antibody is suggested as a starting point. Each investigator may desire to titrate the concentration of antibody and volume of cell lysate in preliminary experiments to determine the optimal conditions. e.g., 1-10 µg/10^7 cells/1 mL lysate. Typically, 2 µg is a sufficient amount of antibody to maximally immunoprecipitate most antigens in 1 mL of extract from 1x10^7 cells. Using as little IP antibody as possible minimizes potential contamination of SDS reduced sample with nonreduced immunoprecipitating antibody light chain. It is not recommended to use more than 10 µg (per mL) or a final of 5 µg per lane.
2. Incubate at 4°C for 1 hour on a rocking platform or a rotator.
3. Add at least 50 µL of pre-equilibrated bead slurry to capture the immune complexes.
4. Incubate for 1 hour or overnight at 4°C on a rocking platform or a rotator.
Note: Step 1 and 3 can combined for a single incubation.
5. Centrifuge the tube at 2,500 xg for 30 seconds at 4°C.
6. Carefully remove supernatant completely and wash the beads 3-5 times with 500 µL of cold Lysis Buffer, centrifuging to pellet beads in between each wash. In order to minimize background, care should be given to remove the supernatant completely after each wash.
7. After the last wash, carefully aspirate supernatant and add 50 µL of 1X Laemmli sample buffer (or any equivalent SDS-PAGE sample loading Buffer) to bead pellet.
Note: Please take into consideration composition of the Loading buffer. Reducing agents can be added.
8. Vortex and heat to 90-100°C for 10 minutes.
9. Centrifuge at 10,000 xg for 5 minutes, collect supernatant carefully and load onto the gel.
10. Alternatively, the supernatant samples can be collected, transferred to clean tube and frozen at -80°C if the gel is to be run later.
11. Follow manufacturer's instructions for SDS-PAGE.

Procedure Step II: Immunoblotting (Western Blotting, WB)
1. Transfer proteins from the gel onto PVDF or nitrocellulose membrane following instructions provided by the transfer system manufacturer for best protein transfer results.
2. Optional: To determine whether the proteins have been transferred to membrane, stain with a 0.1% Ponceau S solution. Protein bands can be visualized after staining for 5 minutes. To remove the Ponceau S stain, rinse with distilled water or TBS-T until most of the dye is removed before moving on to blocking step. Residual dye will not affect subsequent steps.
3. Remove membrane and soak in transfer buffer.
4. Under chemical hood, place the membrane in TrueBlot® Enhancer Solution and soak for 2minutes, then wash with TBS-T.
5. Place the membrane into the 5% TrueBlot Blocker in TrueBlot® Assay Buffer (enough to cover the membrane) and incubate for 2 hours at room temperature or overnight at 4°C on a rocking platform. [Preparation of 5% TrueBlot Blocker in TrueBlot Assay Buffer: Dilute 20X TrueBlot Assay Buffer with dH20 to 1X. Using TrueBlot® Blocker Powder, make a 5% (w/v) solution.]
Note: it is recommended to use Milk as the blocking reagent as BSA does not effectively block the reduced Ig chain recognition.
6. Remove the blocking buffer and rinse blot with TBS-T.
7. Prepare the primary goat immunoblotting antibody in Blocking Buffer as recommended by the supplier. If the recommended concentration is not known use a standard concentration of 1-2 µg/ml. If using hybrid- oma tissue culture supernatant or serum for immunoblotting, preliminary experiments should be performed to evaluate whether dilution of the supernatant or serum is needed for best results.
8. Incubate the blot with primary antibody for at least 2 hours at room temperature of overnight at 4°C on rocking platform.
Note: Shorter times should be determined empirically for optimal results
9. After the overnight incubation of the membrane with the primary antibody, wash the blot at least 3-5 times in TBS-T, each wash for a minimum of 5-10 minutes each. Total should be more than 1 hour.
10. Prepare the secondary antibody Goat IgG TrueBlot® at a 1:1,000 dilution in the Blocking Buffer.
Note: Please avoid the presence of sodium azide in this step as it is deleterious to the HRP enzyme.
11. Incubate the blot with the TrueBlot® secondary antibody for one hour at room temperature on a rocking platform.
12. Wash the blot at least 3-5 times in TBS-T, each wash for at least 5 minutes each. Total should be more than 1 hour.
13. Prepare Substrate: Mix equal volumes of Substrate A and B
14. Incubate the blot in chemiluminescent-HRP substrate working solution (combined A and B) for 1- 0.5 minutes.
15. Expose the blot to X-ray film for an appropriate time period. For best results, expose for ten seconds, one minute, five minutes and 20 minutes.

Buffer: 0.01 M Sodium Phosphate, 0.15 M Sodium Chloride, pH 7.2

Stabilizer: 0.1 mg/mL Bovine Serum Albumin (BSA) - IgG and Protease free, 50 % (v/v) Glycerol