Anti-Vinculin antibody [EPR20407] - Loading Control

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Vinculin antibody [EPR20407] - Loading Control (AB219649)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Vinculin antibody [EPR20407] - Loading Control (AB219649)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Vinculin antibody [EPR20407] - Loading Control (AB219649)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Vinculin antibody [EPR20407] - Loading Control (AB219649)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Vinculin antibody [EPR20407] - Loading Control (AB219649)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling Vinculin with ab219649 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab219649 Anti-Vinculin antibody [EPR20407]was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Vinculin antibody [EPR20407] - Loading Control (AB219649)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Vinculin antibody [EPR20407] - Loading Control (AB219649)

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.

• WB

Supplier Data

Western blot - Anti-Vinculin antibody [EPR20407] - Loading Control (AB219649)

Blocking/Dilution buffer : 5% NFDM/TBST.

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

All lanes:

Western blot - Anti-Vinculin antibody [EPR20407] - Loading Control (ab219649) at 1/1000 dilution

Lane 1:

Human fetal kidney lysate at 20 µg

Lane 2:

Human fetal heart lysate at 20 µg

Lane 3:

Human testis lysate at 20 µg

Lane 4:

Human fetal liver lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/4000 dilution

Predicted band size: 124 kDa

Observed band size: 123 kDa

false

• WB

Supplier Data

Western blot - Anti-Vinculin antibody [EPR20407] - Loading Control (AB219649)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Vinculin antibody [EPR20407] - Loading Control (ab219649) at 1/1000 dilution

Lane 1:

PC-3 (Human prostate adenocarcinoma cell line) whole cell lysate at 20 µg

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 3:

K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 124 kDa

Observed band size: 123 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-Vinculin antibody [EPR20407] - Loading Control (AB219649)

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.

All lanes:

Western blot - Anti-Vinculin antibody [EPR20407] - Loading Control (ab219649) at 1/1000 dilution

Lane 1:

Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

VCL knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human VCL (Vinculin) knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-vcl-vinculin-knockout-a-431-cell-line-ab261898'>ab261898</a>)

Lane 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 4:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Predicted band size: 124 kDa

Observed band size: 124 kDa

false

• WB

Lab

Western blot - Anti-Vinculin antibody [EPR20407] - Loading Control (AB219649)

False colour image of Western blot : Anti-PD-L1 antibody [CAL10] – Mouse IgG1 (Chimeric); Rabbit anti-Vinculin antibody (ab219649) loading control staining at 1/1000 dilution, shown in red. In Western blot, ab279292 was shown to bind specifically to PD-L1. A band was observed at 45 kDa in wild-type A549 cell lysates with no signal observed at this size in Cd274 knockout cell line. To generate this image, wild-type and Cd274 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-PD-L1 antibody [CAL10] - Mouse IgG1 (Chimeric) (<a href='/en-us/products/primary-antibodies/pd-l1-antibody-cal10-mouse-igg1-chimeric-ab279292'>ab279292</a>) at 1/1000 dilution

Lanes 1 - 2:

Western blot - Human wild-type A549 cell line (ab255450)

Lane 1:

Wild-type A549 Control IFN-gamma (0 ng/mL, 48 h) at 20 µg

Lane 2:

Wild-type A549 Treated IFN-gamma (100 ng/mL, 48 h) at 20 µg

Lanes 3 - 4:

Western blot - Human CD274 (PD-L1) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-cd274-pd-l1-knockout-a549-cell-line-ab267055'>ab267055</a>)

Lane 3:

CD274 knockout A549 Control IFN-gamma (0 ng/mL, 48 h) at 20 µg

Lane 4:

CD274 knockout A549 Treated IFN-gamma (100 ng/mL, 48 h) at 20 µg

Secondary

All lanes:

Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution

Predicted band size: 33 kDa

Observed band size: 45 kDa

false

• WB

Supplier Data

Western blot - Anti-Vinculin antibody [EPR20407] - Loading Control (AB219649)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Vinculin antibody [EPR20407] - Loading Control (ab219649) at 1/1000 dilution

Lane 1:

Mouse heart lysate at 10 µg

Lane 2:

Mouse spleen lysate at 10 µg

Lane 3:

Rat heart lysate at 10 µg

Lane 4:

Rat kidney lysate at 10 µg

Lane 5:

Rat spleen lysate at 10 µg

Lane 6:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 7:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 124 kDa

Observed band size: 123 kDa

false

Exposure time: 4s