Vimentin Mouse Monoclonal Antibody [A6-C1]

☑ Relative expression (RE)

Western blot analysis of Vimentin on different lysates with Mouse anti-Vimentin antibody (M1412-1) at 1/2,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: C2C12 cell lysate
Lane 3: L6 cell lysate
Lane 4: Daudi cell lysate (negative)

Lysates/proteins at 20 µg/Lane.

Predicted band size: 54 kDa
Observed band size: 54 kDa

Exposure time: 24 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 (M1412-1) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Knockout (KO)

All lanes: Western blot analysis of Vimentin with anti-Vimentin antibody (M1412-1) at 1:500 dilution.
Lane 1: Wild-type Hela whole cell lysate (10 µg).
Lane 2: Vimentin knockout Hela whole cell lysate (10 µg).

M1412-1 was shown to specifically react with Vimentin in wild-type Hela cells. No band was observed when Vimentin knockout sample was tested. Wild-type and Vimentin knockout samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary antibody (M1412-1, 1:500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG-HRP Secondary Antibody (HA1006) at 1:100,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of HeLa cells labeling Vimentin with Mouse anti-Vimentin antibody (M1412-1) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-Vimentin antibody (M1412-1) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488, HA1125) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

beta Tubulin (ET1602-4, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) were used as the secondary antibody at 1/1,000 dilution.

Immunohistochemical analysis of paraffin-embedded human kidney tissue with Mouse anti-Vimentin antibody (M1412-1) at 1/400 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (M1412-1) at 1/400 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.