- MA5-32041 - Invitrogen Antibodies MLH1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of MLH1 in A431 cell lysate using a MLH1 Monoclonal antibody (Product # MA5-32041) at a dilution of 1:1,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of MLH1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR1027533_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of MLH1 was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) and Hela MLH1 KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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-MLH1 Recombinant Rabbit Monoclonal Antibody (SP08-04) (Product # MA5-32041, 1:1,000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to MLH1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-MLH-1 Rabbit Recombinant Monoclonal Antibody (Product # MA5-32041) and a 84 kDa band corresponding to MLH-1 was observed in cell lines tested except HCT 116 and DU 145 which are reported to be negative. An uncharacterized band at ~150 kDa was observed across the cell lines tested. Modified whole cell extracts (30 µg lysate) of HeLa (Lane 1), A-431 (Lane 2), SW480 (Lane 3), HCT 116 (Lane 4), PC3 (Lane 5), DU 145 (Lane 6) and Raji (Lane 7) were electrophoresed using Novex® 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-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemical analysis of MLH1 in HepG2 cells using a MLH1 Monoclonal antibody (Product # MA5-32041) as seen in green. The nuclear counter stain is DAPI (blue). Cells were fixed in paraformaldehyde, permeabilised with 0.25% Triton X100/PBS.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of MLH1 was performed using 70% confluent log phase SW480 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 1 hour at room temperature. SW480 cells were labeled with MLH1 Rabbit Recombinant Monoclonal Antibody (Product # MA5-32041) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) 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 of SW480 showing nuclear 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)
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Experimental details: Flow Cytometric analysis of MLH1 in Hela cells using a MLH1 Monoclonal Antibody (Product # MA5-32041) at a dilution of 1:50, as seen in red compared with an unlabelled control (cells without incubation with primary antibody; black). Alexa Fluor 488-conjugated goat anti rabbit IgG was used as the secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous MLH1 protein using Anti-MLH1 Antibody: ChIP was performed using Anti-MLH1 Rabbit Recombinant Monoclonal Antibody (Product # MA5-32041, 5 µg) on sheared chromatin from methyl methanesulfonate treated SW480 cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to transcriptional start site and gene body (+2Kb) of GAPDH, ACTB promoter, CDKN1A intron 1 and SATA satellite repeats. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

MA5-32041