Western blot analysis of TIM 3 on different lysates with Mouse anti-TIM 3 antibody (EM1701-19) at 1/2,000 dilution. Lane 1: A549 cell lysate (no heat) Lane 2: HepG2 cell lysate (no heat) Lane 3: HDLM-2 cell lysate Notice: no heat means the lysate is not boiled. Lysates/proteins at 30 µg/Lane. Predicted band size: 33 kDa Observed band size: 42 kDa Exposure time: 3 minutes 10 seconds; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (EM1701-19) at 1/2,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.