ELISA using ab134047 at varying antibody concentrations and antigen concentration at 250 ng/mL. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (1/2500) was used as the secondary antibody.

All lanes : Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/1000 dilution

Lane 1 : HUVEC (Human umbilical vein endothelial cell) whole cell lysate
Lane 2 : HUVEC (Human umbilical vein endothelial cell) treated with 10 ng/ml TNF-a for 16 h, whole cell lysate
Lane 3 : bEnd.3 (Mouse brain endothelioma) whole cell lysate
Lane 4 : bEnd.3 (Mouse brain endothelioma) treated with 10 µg/ml LPS for 24 h, whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 81 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?
Additional bands at: 36 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 2 seconds

Rabbit monoclonal [EPR16891] to GAPDH (ab181602) used as loading control.

Blocking/Diluting buffer: 5% NFDM/TBST.

Stimulation may be required to allow detection of the target protein due to low levels of endogenous expression in some samples.

All lanes : Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/1000 dilution

Lane 1 : Hut-78 (Human Sezary syndrome cutaneous T lymphocyte) whole cell lysate
Lane 2 : SK-OV-3 (Human ovarian cancer epithelial cell) whole cell lysate
Lane 3 : RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 4 : J774A.1 (Mouse reticulum cell sarcoma monocyte macrophage) whole cell lysate
Lane 5 : LADMAC (Mouse bone marrow monocyte macrophage) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 81 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?
Additional bands at: 36 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 7 seconds

Rabbit monoclonal [EPR16891] to GAPDH (ab181602) used as loading control.

Blocking/Diluting buffer: 5% NFDM/TBST.

Immunohistochemical staining of paraffin embedded human tonsil with purified ab134047 at a dilution of 1/500. A pre-diluted HRP polymer for rabbit/mouse IgG was used as the secondary antibody and the sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunofluorescent staining of K562 cells (fixed in 4% PFA, permeabilized with 0.1% Triton X 100) using purified ab134047 at a dilution of 1/250. An Alexa Fluor^® 488 goat anti-rabbit antibody was used as the secondary at a dilution of 1/500 and the cells were counter stained with DAPI. The negative control is shown in the bottom right hand panel - for the negative control, Alex Fluor^® 594 goat anti-mouse was used at a dilution of 1/500.

Flow Cytometry analysis of K562 (human chronic myelogenous leukemia) cells labeling VCAM1 with purified ab134047 at 1/40 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^®488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

All lanes : Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/10000 dilution (purified)

Lane 1 : Mouse kidney
Lane 2 : Rat spleen

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 81 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

VCAM1 was immunoprecipitated from Human fetal liver lysate with ab134047 at 1/110 dilution.

Western blot was performed from the immunoprecipitate using ab134047 at 1/1000 dilution.

VeriBlot for IP secondary antibody (Peroxidase conjugated),was used as secondary antibody at 1/1000 dilution.

Lane 1: Human fetal liver lysate.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 1 second.

IHC image of VCAM1 staining in Human spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with unpurified ab134047, 1/200 dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/2000 dilution (purified) + Rat kidney tissue lysate at 20 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 81 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST.

Dilution buffer: 5% NFDM/TBST.

Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/10000 dilution (purified) + NIH/3T3 cell lysate at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 81 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST.

Dilution buffer: 5% NFDM/TBST.

Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/10000 dilution (purified) + Human fetal liver tissue lysate at 20 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 81 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST.

Dilution buffer: 5% NFDM/TBST.

Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/10000 dilution (purified) + HuT-78 cell lysate at 10 µg

Secondary
HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 81 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST.

Dilution buffer: 5% NFDM/TBST.

IHC image of VCAM1 staining in Mouse spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with unpurified ab134047, 1/200 dilution, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

All lanes : Anti-VCAM1 antibody [EPR5047] (ab134047) at 1/1000 dilution (unpurified)

Lane 1 : Human fetal liver tissue lysate
Lane 2 : HuT 78 cell lysate
Lane 3 : NIH/3T3 cell lysate
Lane 4 : Mouse brain tissue lysate
Lane 5 : Mouse kidney tissue lysate
Lane 6 : Mouse spleen tissue lysate
Lane 7 : Rat brain tissue lysate
Lane 8 : Rat kidney tissue lysate
Lane 9 : Rat spleen tissue lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/2000 dilution

Predicted band size: 81 kDa