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89477
Bax (2D2) Mouse mAb
Primary Antibodies
Monoclonal Antibody

Bax (2D2) Mouse mAb #89477

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  1. WB
  2. IP
  3. IF
Western Blotting Image 1: Bax (2D2) Mouse mAb

Western blot analysis of extracts from various cell lines using Bax (2D2) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). As expected, DU 145 cells are negative for Bax expression.

Immunoprecipitation Image 1: Bax (2D2) Mouse mAb

Immunoprecipitation of Bax protein from HT-1080 cell extracts. Lane 1 is 10% input, lane 2 is Mouse (G3A1) mAb IgG1 Isotype Control #5415, and lane 3 is Bax (2D2) Mouse mAb. Western blot analysis was performed using Bax (2D2) Mouse mAb. An Anti-Mouse Ig HRP conjugated secondary antibody was used to mask the heavy and light chain signals.

Immunofluorescence Image 1: Bax (2D2) Mouse mAb

Confocal immunofluorescent analysis of untreated MCF7 cells (left, Bax-inactive), or MCF7 cells (center, Bax-activated) and DU 145 cells (right, Bax-deficient) irradiated with UV (200 mJ/cm2) then recovered for 2 hr in 20 µM Z-VAD, using Bax (2D2) Mouse mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue). Staining was achieved with a modified CHAPS protocol (see product webpage).

To Purchase # 89477S
製品番号 サイズ 価格 在庫
89477S
100 µl

Supporting Data

REACTIVITY H
SENSITIVITY Endogenous
MW (kDa) 20
Source/Isotype Mouse IgG1

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Usage Information

Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:100
Immunofluorescence (Immunocytochemistry) 1:6400

Storage

Supplied in 10 mM PBS containing 0.05% BSA and 0.05% sodium azide. Stable for 24 months when stored at -20°C. This product will freeze at -20°C so it is recommended to aliquot into single-use vials to avoid multiple freeze/thaw cycles. A slight precipitate may be present, but will not interfere with antibody performance.

Protocol

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Western Blotting Protocol

For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

NOTE: Please refer to primary antibody product webpage for recommended antibody dilution.

A. Solutions and Reagents

From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit

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

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 10X Tris Buffered Saline (TBS): (#12498) To prepare 1 L 1X TBS: add 100 ml 10X to 900 ml dH2O, mix.
  3. 1X 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. Dilute to 1X with dH2O.
  4. 10X Tris-Glycine SDS Running Buffer: (#4050) To prepare 1 L 1X running buffer: add 100 ml 10X running buffer to 900 ml dH2O, mix.
  5. 10X Tris-Glycine Transfer Buffer: (#12539) To prepare 1 L 1X Transfer Buffer: add 100 ml 10X Transfer Buffer to 200 ml methanol + 700 ml dH2O, mix.
  6. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2O, mix.
  7. Nonfat Dry Milk: (#9999).
  8. Blocking Buffer: 1X TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  9. Wash Buffer: (#9997) 1X TBST.
  10. Primary Antibody Dilution Buffer: 1X TBST with 5% nonfat dry milk; for 20 ml, add 1.0 g nonfat dry milk to 20 ml 1X TBST and mix well.
  11. Biotinylated Protein Ladder Detection Pack: (#7727).
  12. Prestained Protein Marker, Broad Range (11-190 kDa): (#13953).
  13. Blotting Membrane and Paper: (#12369) This protocol has been optimized for nitrocellulose membranes. Pore size 0.2 µm is generally recommended.
  14. Secondary Antibody Conjugated to HRP: Anti-mouse IgG, HRP-linked Antibody (#7076).
  15. Detection Reagent: SignalFire™ ECL Reagent (#6883).

B. Protein Blotting

A general protocol for sample preparation.

  1. Treat cells by adding fresh media containing regulator for desired time.
  2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
  3. Lyse cells by adding 1X SDS sample buffer (100 µl per well of 6-well plate or 500 µl for a 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
  4. Sonicate for 10–15 sec to complete cell lysis and shear DNA (to reduce sample viscosity).
  5. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice.
  6. Microcentrifuge for 5 min.
  7. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).

    NOTE: Loading of prestained molecular weight markers (#13953, 5 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended.

  8. Electrotransfer to nitrocellulose membrane (#12369).

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.

I. Membrane Blocking

  1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 min at room temperature.
  2. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature.
  3. Wash three times for 5 min each with 15 ml of TBST.

II. Primary Antibody Incubation

  1. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the product webpage) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
  2. Wash three times for 5 min each with 15 ml of TBST.
  3. Incubate membrane with Anti-mouse IgG, HRP-linked Antibody (#7076 at 1:2000) and Anti-biotin, HRP-linked Antibody (#7075 at 1:1000–1:3000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  4. Wash three times for 5 min each with 15 ml of TBST.
  5. Proceed with detection (Section D).

D. Detection of Proteins

Directions for Use:

  1. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST.
  2. Prepare 1X SignalFire™ ECL Reagent (#6883) by diluting one part 2X Reagent A and one part 2X Reagent B (e.g. for 10 ml, add 5 ml Reagent A and 5 ml Reagent B). Mix well.
  3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

* Avoid repeated exposure to skin.

posted June 2005

revised June 2020

Protocol Id: 19

Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein G 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 G Magnetic Beads: (#70024).
  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 (Highly Recommended)

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

  1. Briefly vortex the stock tube to resuspend the magnetic beads.
  2. IMPORTANT: Pre-wash #70024 magnetic beads just prior to use:

  3. 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.

  4. 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.

  5. Incubate with rotation for 20 minutes at room temperature.
  6. 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.
  7. 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, and Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal 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. Load the sample (15-30 µl) on SDS-PAGE.
  5. Analyze sample by western blot (see Western Immunoblotting Protocol).

NOTE: For proteins with molecular weights in the range of around 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) as a secondary antibody to minimize interference produced by denatured heavy chains. For proteins with molecular weights in the range of around 25 kDa, Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127) is recommended to minimize interference produced by denatured light chains.

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.

posted December 2008

revised April 2018

Protocol Id: 121

Immunofluorescence (Immunocytochemistry), CHAPS Protocol

IMPORTANT: This protocol employs an atypical permeabilization strategy with which only certain targets are compatible. Triton X-100 is removed from all buffers and replaced with a brief incubation in 0.2% CHAPS detergent. Where appropriate, this protocol will be linked to its validated antibody under the Product Information banner on the product-specific webpage.

A. Solutions and Reagents

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

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1L 1X PBS, add 100 ml 10X Wash Buffer, Phosphate Buffered Saline (#12528) to 900 ml dH2O, mix. Adjust pH to 8.0.
  2. 4% Formaldehyde, Methanol-Free (#47746): Use fresh.
  3. CHAPS Permeabilization Buffer (1X PBS / 0.2% CHAPS detergent): To prepare 10 ml, add 0.02g CHAPS detergent to 10 ml 1X PBS and mix well.
  4. Detergent-free Blocking Buffer (1X PBS / 5% normal serum): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) to 9.5 mL 1X PBS, mix well. Store at 4°C.
  5. Detergent-free Antibody Dilution Buffer (1X PBS / 1% BSA): To prepare 10 ml, add 0.1 g BSA (#9998) to 10 ml 1X PBS and mix well. Store at 4°C.
  6. Fluorochrome-conjugated Secondary Antibody: Use a secondary antibody that is reactive to your primary antibody host species (e.g., rabbit). Click here for a list of secondary antibodies approved for immunofluorescence.

B. Fixation and Permeabilization

    NOTE: All subsequent incubations should be carried out at room temperature (20-25°C) unless noted otherwise.

  1. Aspirate culture media then cover cells to a depth of 2–3 mm with 4% formaldehyde.
  2. Allow cells to fix for 15 min.
  3. Rinse three times in 1X PBS for 5 min each.
  4. Aspirate PBS then incubate fixed cells in CHAPS Permeabilization Buffer for 10 min.
  5. Rinse three times in 1X PBS for 5 min each.

C. Immunostaining

  1. Block specimen in Detergent-free Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody in Detergent-free Antibody Dilution Buffer (see product website for recommended dilution range).
  3. Aspirate blocking solution then apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.
  6. NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Section C, Step 8.

  7. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Detergent Free Antibody Dilution Buffer for 1–2 hr protected from light.
  8. Rinse three times in 1X PBS for 5 min each protected from light.
  9. Counterstain as appropriate.
  10. NOTE: When including fluorescent cellular dyes in your experiment (DNA dyes, etc.), please refer to the dye product page for its recommended protocol. View our listing of cellular dyes validated for use in immunofluorescence.

  11. Mount samples for imaging.
  12. For long-term storage, store samples at 4°C protected from light.

posted June 2020

Protocol Id: 2049

Specificity / Sensitivity

Bax (2D2) Mouse mAb recognizes endogenous levels of total Bax protein.

Species Reactivity:

Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the N-terminal region of human Bax protein.

Background

Bax is a key component for cellular induced apoptosis through mitochondrial stress (1). Upon apoptotic stimulation, Bax forms oligomers and translocates from the cytosol to the mitochondrial membrane (2). Through interactions with pore proteins on the mitochondrial membrane, Bax increases the membrane's permeability, which leads to the release of cytochrome c from mitochondria, activation of caspase-9 and initiation of the caspase activation pathway for apoptosis (3,4).

  1. Wei, M.C. et al. (2001) Science 292, 727-730.
  2. Jürgensmeier, J.M. et al. (1998) Proc Natl Acad Sci U S A 95, 4997-5002.
  3. Narita, M. et al. (1998) Proc Natl Acad Sci U S A 95, 14681-6.
  4. Marzo, I. et al. (1998) Science 281, 2027-31.

Pathways & Proteins

Explore pathways + proteins related to this product.

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For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DyLight is a trademark of Thermo Fisher Scientific, Inc. and its subsidiaries.
ProLong is a registered trademark of Life Technologies Corporation.
Tween is a registered trademark of ICI Americas, Inc.
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