Western blot was performed using Anti-MLH-1 Rabbit Polyclonal Antibody (Product # PA5-78062) and a 84 kDa band corresponding to MLH-1 was observed in all the cell lines tested except HCT 116 and DU 145 which are reported to be negative. Modified whole cell extracts (1% SDS) (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 (1:500 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).
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) andHela MLH1 KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 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 Anti-MLH1 Polyclonal Antibody (Product # PA5-78062, 1:500 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:8,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (Product # A38556). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to MLH1.Uncharacterized band was observed in all the samples at 60 kDa.
Supplier Page from Thermo Fisher Scientific for MLH1 Antibody