B7-H6 (E9Q7L) Rabbit mAb #20617

Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS, add 50 ml 20X PBS to 950 ml dH₂O, mix.

2. 10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH₂O, mix.

   NOTE: Add 1 mM PMSF (#8553) immediately prior to use.

3. 3X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.
4. Protein A Magnetic Beads: (#73778).
5. Magnetic Separation Rack: (#7017) or (#14654).
6. 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 900 µl dH₂O, mix.
7. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer.

B. Preparing Cell Lysates

1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
3. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min.
4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
5. Sonicate on ice three times for 5 sec each.
6. Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C.

C. Immunoprecipitation

Cell Lysate Pre-Clearing (Optional)

A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.

1. Briefly vortex the stock tube to resuspend the magnetic beads.

   IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:

2. Transfer 20 μl of bead slurry to a clean tube. Place the tube in a magnetic separation rack for 10-15 seconds.

   Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.

3. Add 200 μl cell lysate to 20 μl of pre-washed magnetic beads.

   IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.

4. Incubate with rotation for 20 minutes at room temperature.
5. Separate the beads from the lysate using a magnetic separation rack, transfer the pre-cleared lysate to a clean tube, and discard the magnetic bead pellet.
6. Proceed to immunoprecipitation section.

Immunoprecipitation

IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP^® Isotype Control #3900 for rabbit monoclonal primary antibodies, Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal IgG1 primary antibodies, Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656 for mouse monoclonal IgG2a primary antibodies, Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 for mouse monoclonal IgG2b primary antibodies, and Mouse (E1D5H) mAb IgG3 Isotype Control #37988 for mouse monoclonal IgG3 primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples.

1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate. Incubate with rotation overnight at 4°C. to form the immunocomplex.
2. Pre-wash magnetic beads (see Cell Lysate Pre-Clearing section, steps 1 and 2).
3. Transfer the lysate and antibody (immunocomplex) solution to the tube containing the pre-washed magnetic bead pellet.
4. Incubate with rotation for 20 min at room temperature.
5. Pellet beads using magnetic separation rack. Wash pellets five times with 500 μl of 1X cell lysis buffer. Keep on ice between washes.
6. Proceed to analyze by western immunoblotting or kinase activity (section D).

D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting

1. Resuspend the pellet with 20-40 µl 3X SDS sample buffer, briefly vortex to mix, and briefly microcentrifuge to pellet the sample.
2. Heat the sample to 95-100°C for 5 min.
3. Pellet beads using magnetic separation rack. Transfer the supernatant to a new tube. The supernatant is the sample.
4. Analyze sample by western blot (see Western Immunoblotting Protocol).

NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).

For Analysis by Kinase Assay

1. Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice.
2. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate.
3. Incubate for 30 min at 30°C.
4. Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
5. Transfer supernatant containing phosphorylated substrate to another tube.
6. Heat the sample to 95-100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
7. Load the sample (15-30 µl) on SDS-PAGE gel.

Flow Cytometry, Methanol Permeabilization Protocol for Rabbit Antibodies

A. Solutions and Reagents

All reagents required for this protocol may be efficiently purchased together in our Intracellular Flow Cytometry Kit (Methanol) #13593, or individually using the catalog numbers listed below.

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water mix.
2. 4% Formaldehyde, Methanol-Free (#47746)
3. 100% Methanol (#13604): Chill before use
4. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
5. Recommended Anti-Rabbit secondary antibodies::
   • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor^® 488 Conjugate) #4412
   • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor^® 594 Conjugate) #8889
   • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor^® 647 Conjugate) #4414
   • Anti-Rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #79408

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.

B. Fixation

NOTE: Adherent cells or tissue should be dissociated and in single-cell suspension prior to fixation.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

NOTE: If using whole blood, lyse red blood cells and wash by centrifugation prior to fixation.

NOTE: Antibodies targeting CD markers or other extracellular proteins may be added prior to fixation if the epitope is disrupted by formaldehyde and/or methanol. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. However, note that some fluorophores (including PE and APC) are damaged by methanol and thus should not be added prior to permeabilization. Conduct a small-scale experiment if you are unsure.

1. Pellet cells by centrifugation and remove supernatant.
2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
3. Fix for 15 min at room temperature (20-25°C).
4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 0.5-1 ml 1X PBS. Proceed to Permeabilization step.
   1. Alternatively, cells may be stored overnight at 4°C in 1X PBS.

C. Permeabilization

1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
2. Permeabilize for a minimum of 10 min on ice.
3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

1. Aliquot desired number of cells into tubes or wells. (Generally, 5x10⁵ to 1x10⁶ cells per assay.)
2. Wash cells by centrifugation in excess 1X PBS to remove methanol. Discard supernatant in appropriate waste container. Repeat if necessary.
3. Resuspend cells in 100 µl of diluted primary antibody, prepared in Antibody Dilution Buffer at a recommended dilution or as determined via titration.
4. Incubate for 1 hr at room temperature.
5. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
6. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in Antibody Dilution Buffer at the recommended dilution).
7. Incubate for 30 min at room temperature. Protect from light.
8. Wash by centrifugation in Antibody Dilution Buffer or 1X PBS. Discard supernatant. Repeat.
9. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

非偶联兔抗体的流式细胞术（活细胞）实验步骤

A. 溶液与试剂

注：使用反渗透去离子水 (RODI) 或同等级别的水配制溶液。

1. 1X 磷酸盐缓冲生理盐水 (PBS)：若要制备 1 L 1X PBS：将 100 ml 10X PBS (#12528) 添加到 900 ml 水，混合。
2. 抗体稀释缓冲液：购买即用型流式细胞术抗体稀释缓冲液 (#13616)，或在 100 ml 1x PBS 中溶解 0.5 g 牛血清白蛋白 (BSA) (#9998) 来配制 0.5 % BSA PBS 缓冲液。4°C 保存。
3. 建议使用抗兔二抗：
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 488 Conjugate) #4412
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 594 Conjugate) #8889
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (Alexa Fluor^® 647 Conjugate) #4414
   • Anti-Rabbit IgG (H+L), F(ab')₂ Fragment (PE Conjugate) #79408

注：在您的实验中加入荧光细胞染料（包括活力指示染料、DNA 染料等）时，请参考染料产品网页，了解建议的实验步骤。访问 www.cellsignal.com，了解经验证用于流式细胞术的细胞染料完整列表。

B. 免疫染色

注：使用血细胞计数器或备选方法计数细胞。

注：如果使用全血，则需裂解红血细胞，并在免疫染色之前通过离心分离洗涤。

注：人 Fc 受体与兔 IgG 发生交叉反应。当感兴趣的细胞表达高水平的 Fc 受体蛋白（例如，巨噬细胞/单核细胞谱系）时，在用兔抗体进行免疫染色之前，用人 Fc 块预孵育活细胞。

注：最佳离心条件会根据细胞类型和试剂容量变动。一般，1-5 分钟 150-300g 将足以使细胞沉淀下来。

1. 将所需数目的细胞分装入试管或小孔中。（通常，每次测定 5 x 10⁵ 至 1 x 10⁶ 个细胞。）
2. 通过离心使细胞沉淀下来，移除上清液。
3. 在 100 µl 稀释的一抗中重悬细胞，这种一抗按建议的稀释度或如通过滴定所确定那样以抗体稀释缓冲液配制。
4. 在冰上孵育 30 分钟至 1 小时。避光。
5. 在抗体稀释缓冲液或 1X PBS 中通过离心洗涤。弃去上清液。重复。
6. 在 100 µl 稀释的荧光物质偶联的二抗（按建议稀释度用抗体稀释缓冲液制备）中重悬细胞。
7. 在冰上孵育 30 分钟。避光。
8. 用抗体稀释缓冲液进行离心洗涤。弃去上清液。重复。
9. 在 200-500 μl 抗体稀释缓冲液中重悬细胞后用流式细胞分析仪进行分析。