All lanes : Anti-Vinculin antibody [EPR20407] (ab219649) at 1/1000 dilution

Lane 1 : Wild-type A431 whole cell lysate
Lane 2 : VCL knockout A431 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : Jurkat whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 124 kDa
Observed band size: 124 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab219649 observed at 124 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab219649 was shown to react with VCL in A431 wild-type cells in Western blot. Loss of signal was observed when VCL knockout sample was used. A431 wild-type and VCL knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween®) before incubation with ab219649 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human breast tissue labeling Vinculin with ab219649 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Membranous staining on myoepithelium of human breast is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Vinculin with ab219649 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Membranous and cytoplasmic staining on smooth muscle cells of human colon is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-Vinculin antibody [EPR20407] (ab219649) at 1/1000 dilution

Lane 1 : PC-3 (Human prostate adenocarcinoma cell line) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 124 kDa
Observed band size: 123 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Vinculin antibody [EPR20407] (ab219649) at 1/1000 dilution

Lane 1 : Human fetal kidney lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human testis lysate
Lane 4 : Human fetal liver lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution

Predicted band size: 124 kDa
Observed band size: 123 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1-3: 15 seconds; Lane 4: 30 seconds.

All lanes : Anti-Vinculin antibody [EPR20407] (ab219649) at 1/1000 dilution

Lane 1 : Mouse heart lysate
Lane 2 : Mouse spleen lysate
Lane 3 : Rat heart lysate
Lane 4 : Rat kidney lysate
Lane 5 : Rat spleen lysate
Lane 6 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 7 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 124 kDa
Observed band size: 123 kDa why is the actual band size different from the predicted?


Exposure time: 4 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human gastric cancer tissue labeling Vinculin with ab219649 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Membranous and cytoplasmic staining on smooth muscle cells of human gastric cancer is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human prostate cancer tissue labeling Vinculin with ab219649 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Membranous and cytoplasmic staining on smooth muscle cells of human prostate cancer is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Vinculin with ab219649 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Membranous and cytoplasmic staining on smooth muscle cells of mouse colon is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat stomach tissue labeling Vinculin with ab219649 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Membranous and cytoplasmic staining on smooth muscle cells of rat stomach is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.