MA1-153

antibody from Invitrogen Antibodies

Targeting: BCR ALL, BCR1, CML, D22S11, D22S662, PHL

Provider product page for MA1-153

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [1]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [2]
Immunohistochemistry [3]
Other assay [2]

Submit

Product number: MA1-153 - Provider product page
Provider: Invitrogen Antibodies
Product name: BCR-ABL Monoclonal Antibody (7C6)
Antibody type: Monoclonal
Antigen: Other
Description: Western blot analysis of MA1-153 detects ~130-160 kDa and a 210-250 kDa protein in K562 cells. In addition to BCR-ABL, BCR and ABL are also expressed in these cells and full length BCR is also detected by the antibody. For detection purposes, non-reducing conditions resulted in enhanced detection of BCR-ABL.
Reactivity: Human, Mouse
Host: Mouse
Isotype: IgG
Antibody clone number: 7C6
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: -20°C

BCR protein structure - MA1-153 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of BCR-ABL and BCR was performed following immunoprecipitation from K562 cell lysates using a phosphotyrosine monoclonal antibody for immune complex capture, and a BCR antibody (Product # MA1-153) for Western Blot detection. Immunoprecipitated proteins from 500 µg whole cell lysate and 10 µl of PageRuler Prestained Protein Ladder (Product # 26616) were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a BCR monoclonal antibody (Product # MA1-153) at a dilution of 1:1000 overnight rotating at 4°C, washed in TBST, and probed with a goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:20,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080). Note: Best results were obtained using non-reducing LDS sample buffer in SDS-polyacrylamide gel electrophoresis (Product # 84788).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of BCR was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR981967_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of BCR was performed by loading 30 µg of A-431 Wild Type (Lane 1), A-431 Cas9 (Lane 2) andA-431 BCR KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ 3-8% Tris-Acetate Protein Gel (Product # EA0378BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-BCR-ABL Monoclonal Antibody (7C6) (Product # MA1-153, 1:1,000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:5,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to BCR.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on membrane enriched extracts of K-562 (Lane 1), AML-193 (Lane 2), Jurkat (Lane 3) and Hep G2 (Lane 4). The blot was probed with Anti-BCR-ABL Monoclonal Antibody (Product # MA1-153, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/ml, 1:4000 dilution). A 210 kDa band corresponding to BCR-ABL was detected in K-562 cell line when compared to other cell lines which were reported to be negative. Also the antibody detects 130, 160 kDa band corresponding to BCR across all the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of BCR-ABL (green) in K562 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature. Cells were blocked with 1% Blocker BSA (Product # 37525) for 15 minutes at room temperature. Cells were probed with a BCR-ABL monoclonal antibody (Product # MA1-153) at a dilution of 1:50 for at least 1 hour at room temperature, washed with PBS, and incubated with a DyLight 488-conjugated goat anti-mouse IgG secondary antibody (Product # 35502). Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of BCR-ABL was performed using 70% confluent log phase K-562 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with BCR-ABL Monoclonal Antibody (7C6) (Product # MA1-153) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415). Panel d represents the merged image showing membranous localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of BCR-ABL showing staining in the nucleus of paraffin-embedded human breast carcinoma (right) compared with a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a BCR-ABL monoclonal antibody (Product # MA1-153) diluted in 3% BSA-PBS at a dilution of 1:200 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of BCR-ABL showing staining in the nucleus of paraffin-embedded human hepatocarcinoma (right) compared with a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a BCR-ABL monoclonal antibody (Product # MA1-153) diluted in 3% BSA-PBS at a dilution of 1:200 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of BCR-ABL showing staining in the nucleus and cytoplasm of paraffin-embedded mouse liver tissue (right) compared with a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a BCR-ABL monoclonal antibody (Product # MA1-153) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of BCR-ABL and BCR was performed using K562 whole cell lysate. Antigen-antibody complexes were formed by incubating 800 µg of lysate with 5 µg of a BCR monoclonal antibody (Product # MA1-153) overnight on a rocking platform at 4øC. The immune complexes were captured on 50 µl Protein A/G Agarose (Product # 20421), washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer (Product # 39000). The sample was resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBS-0.1%Tween for 1 hour. The membrane was probed with a phosphotyrosine monoclonal antibody (detecting p-Tyr-BCR-ABL/p-Tyr-BCR) at a dilution of 1:1000 overnight rotating at 4øC, washed in TBST, and probed with a goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:20,000 for 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2. The TF1-BAP model displays similar features as chronic myeloid leukemia (CML) persistent cells. (A) Strategy for generating TF1-BA and TF1-BAP cell lines. (B) Representative images of BMPR1B staining of TF1-BA and TF1-BAP cells. (C) Western blot analysis showing BMPR1B levels in TF1-BA and TF1-BAP cells; Scatter plot showing BMPR1B (GAPDH used as an internal control) n=5. (D) Representative FACS plots of TF1-BA and TF1-BAP cells analyzed for their CD34 and CD38 content. (E) Dot plot showing colony forming cell (CFC) activity from TF1-BA and TF1-BAP cells. (F) Dot plot showing long-term culture-initiating cell (LTC-IC) activity from TF1-BA and TF1-BAP cells. (G) Western blot analysis showing BCR-Abl and P-CRKL levels in TF1-BA and TF1-BAP cells. Bar graph showing BCR-Abl and P-CRKL (GAPDH used as an internal control); n=4. (H) TF1-BA and TF1-BAP cells were analyzed for their content in P-Smad1/5/8 and P-Stat3. Dot plots represent percentage of PSmad1/ 5/8 (left panel), P-Stat3 (middle panel) or double positive P-Smad1/5/8-P-Stat3 (right panel). (E-H) Unpaired t -test significant: * P