Western blot was performed using Anti-MEIS1 Polyclonal Antibody (Product # PA5-19050) and ~55, ~50 kDa bands corresponding to MEIS1 were observed in IMR-32. SK-N-SH and HeLa cell lines but not in Daudi and A-431 which are reported to be negative. Modified Whole cell extracts (1% SDS) (30 µg lysate) of IMR-32 (Lane 1), SK-N-SH (Lane 2), HeLa (Lane 3), Daudi (Lane 4) and A-431 (Lane 5) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0302BOX). 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 (0.1 µg/mL) and detected by chemiluminescence with Rabbit anti-Goat IgG Heavy Chain, Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27014, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
Immunofluorescence analysis of MEIS1 was performed using 70% confluent log phase IMR-32 and A-431 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. The cells were labeled with MEIS1 Rabbit Polyclonal Antibody (Product # PA5-19050) at 1:250 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Rabbit anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Product # A-11078) 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. Panel e represents merged image for negative staining of MEIS1 in A431 cells. Panel f represents control cells with no primary antibody to assess background.
Supplier Page from Thermo Fisher Scientific for MEIS1 Antibody