Epithelial-Mesenchymal Transition (EMT) Antibody Sampler Kit

Western blot analysis of Vimentin on different lysates with Rabbit anti-Vimentin antibody (ET1610-39) at 1/20,000 dilution and competitor's antibody at 1/1,000 dilution.

Lane 1: HeLa cell lysate (10 µg/Lane)
Lane 2: HEK-293 cell lysate (10 µg/Lane)
Lane 3: Jurkat cell lysate (10 µg/Lane)
Lane 4: C2C12 cell lysate (10 µg/Lane)
Lane 5: RAW264.7 cell lysate (10 µg/Lane)
Lane 6: C6 cell lysate (10 µg/Lane)

Predicted band size: 54 kDa
Observed band size: 54 kDa
Exposure time: 14 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 (ET1610-39) at 1/20,000 dilution and competitor's antibody at 1/1,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:50,000 dilution was used for 1 hour at room temperature.

Western blot analysis of N Cadherin on different lysates with Rabbit anti-N Cadherin antibody (ET1607-37) at 1/5,000 dilution and competitor's antibody at 1/1,000 dilution.

Lane 1: 293T cell lysate
Lane 2: A549 cell lysate
Lane 3: HeLa cell lysate
Lane 4: A-172 cell lysate
Lane 5: MCF7 cell lysate (negative)
Lane 6: C2C12 cell lysate
Lane 7: C6 cell lysate

Lysates/proteins at 15 µg/Lane.
Predicted band size: 100 kDa
Observed band size: 140-150 kDa
Exposure time: 2 minutes 6 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 (ET1607-37) at 1/5,000 dilution and competitor's antibody at 1/1,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Western blot analysis of E-Cadherin on different lysates with Rabbit anti-E-Cadherin antibody (HA723564) at 1/5,000 dilution.

Lane 1: 4T1 cell lysate (20 µg/Lane)
Lane 2: C2C12 cell lysate (negative) (20 µg/Lane)
Lane 3: Mouse small intestine tissue lysate (30 µg/Lane)
Lane 4: Mouse colon tissue lysate (30 µg/Lane)
Lane 5: Mouse pancreas tissue lysate (30 µg/Lane)
Lane 6: Rat pancreas tissue lysate (30 µg/Lane)
Lane 7: Rat lung tissue lysate (30 µg/Lane)
Lane 8: Rat colon tissue lysate (30 µg/Lane)
Lane 9: MCF7 cell lysate (20 µg/Lane)
Lane 10: MDA-MB-231 cell lysate (negative) (20 µg/Lane)
Lane 11: HT-29 cell lysate (20 µg/Lane)
Lane 12: Caco-2 cell lysate (20 µg/Lane)

Predicted band size: 98 kDa
Observed band size: 75-130 kDa

Exposure time: Lane 1-5: 10 seconds; Lane 3: 1 minute 16 seconds; ECL: K1801;

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 (HA723564) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Application: IHC-Fr

Species: Mouse

Site: colon

Sample: Frozen section

Antibody concentration: 1/500

Antigen retrieval: Not required

Application: IHC-Fr

Species: Rat

Site: colon

Sample: Frozen section

Antibody concentration: 1/500

Antigen retrieval: Not required

Immunohistochemical analysis of paraffin-embedded mouse colon tissue with Rabbit anti-E-Cadherin antibody (HA723564) at 1/5,000 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 (HA723564) at 1/5,000 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.

Immunocytochemistry analysis of 4T1 (positive) and C2C12 (negative) labeling E-Cadherin with Rabbit anti-E-Cadherin antibody (HA723564) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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 Rabbit anti-E-Cadherin antibody (HA723564) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) 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 (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

Flow cytometric analysis of C2C12 (left, negative) and 4T1 (right, positive) cells labeling E-Cadherin.

Cells were washed twice with cold PBS and resuspend. Then stained with the primary antibody (HA723564, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

E-Cadherin was immunoprecipitated from 0.2 mg 4T1 cell lysate with HA723564 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA723564 at 1/5,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: 4T1 cell lysate (input)
Lane 2: HA723564 IP in 4T1 cell lysate
Lane 3: Rabbit IgG instead of HA723564 in 4T1 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 46 seconds; ECL: K1801