MA5-14847

antibody from Invitrogen Antibodies

Targeting: MET DFNB97, HGFR, RCCP2

Antibody data

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Validation data

MET protein structure - MA5-14847 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CRISPR-Cas9 mediated genome editing ofc-MET (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR752450_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of c-MET was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) and HeLa Cas9 cells transduced with c-MET Lentiviral sgRNA (Lane 3) membrane enriched 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-c-Met Monoclonal Antibody (T.848.5) (Product # MA5-14847) using 1:4,000 dilution and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177 1:8,000 dilution). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). A reduced signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific toc-MET (Fig (b)). Uncharacterized bands was observed in HeLa Cas9 and HeLa Cas9 cells transduced with c-MET Lentiviral sgRNA samples at 39 kDa and 110 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Hepatocyte growth factor receptor was achieved by transfecting MDA-MB-231 with Hepatocyte growth factor receptor specific siRNAs (Silencer® select Product # S8701, S8700). Western blot analysis (Fig. a) was performed using Whole cell extracts from the Hepatocyte growth factor receptor knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with c-Met Monoclonal Antibody (T.848.5) (Product # MA5-14847, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to Hepatocyte growth factor receptor. An uncharacterized band of ~110 kDa was also observed across the cell lines.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-c-Met Monoclonal Antibody (T.848.5) (Product # MA5-14847) and a 160 kDa band corresponding to Hepatocyte growth factor receptor was observed in MDA-MB-231 and not in any other breast cancer cell lines like SK-BR-3, MCF7 and T-47D which are reported as negative. Whole cell extracts (30 µg lysate) of MDA-MB-231 (Lane 1), SK-BR-3 (Lane 2), MCF7 (Lane 3) and T-47D (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). An uncharacterized band of ~110 kDa was also observed across the cell lines.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Hepatocyte growth factor receptor was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with c-Met Monoclonal Antibody (T.848.5) (Product # MA5-14847) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000 dilution), 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: Blue) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Plasma membrane 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: Immunofluorescence analysis of Hepatocyte growth factor receptor was performed using 70% confluent log phase MDA-MB-231 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with c-Met Monoclonal Antibody (T.848.5) (Product # MA5-14847) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000 dilution), 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, 1:300). Panel d represents the merged image showing Nuclear localization. Panel e represents SK-BR-3 cells having no expression of c-Met. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification (DOI: 10.1093/carcin/bgp080).