All lanes : Anti-Vimentin antibody - Cytoskeleton Marker (ab45939) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole cell lysate
Lane 4 : A549 (Human lung adenocarcinoma epithelial cell line) Whole cell lysate
Lane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 6 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
Lane 7 : HUVEC (Human umbilical vein endothelial cell line) Whole cell lysate
Lane 8 : A431 (Human epithelial carcinoma cell line) Whole cell lysate
Lane 9 : Daudi (Human B lymphoblast) Whole cell lysate
Lane 10 : Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : IRDye® 800CW Goat Anti-Rabbit at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 54 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab45939 overnight at 4°C. The membrane was then blocked for an hour using LI-COR® blocking buffer before being incubated with ab92547 overnight at 4°C. Antibody binding was detected using the IRDye® 800CW Goat Anti-Rabbit secondary at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Odyssey® CLx Imaging System.

ab45939 staining Vimentin in wild-type HAP1 cells (top panel) and Vimentin knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab45939 at 1μg/ml dilution and ab195889 at 1/250 dilution (shown in pseudo-color red) overnight at +4°C. The cells were then incubated with ab150081 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488)) at 1/1000 dilution for 1 hour. Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab45939 staining Vimentin in HeLa cells. The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab45939 at 1μg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control, at 1/1000 dilution. Cells were then incubated with ab150120, Goat polyclonal Secondary Antibody to Mouse at 1/1000 dilution (shown in pseudocolour red) and ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG at 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

IHC image of ab45939 staining Vimentin in human kidney formalin fixed paraffin embedded tissue sections*, performed on a Leica Bond. 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 ab45939, 0.1μg/ml working concentration, 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. No primary antibody was used in the secondary only control (shown on the inset).
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.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

ab45939 staining Vimentin in SV40LT-SMC cells. The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab45939 at 1μg/ml (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin - Microtubule Marker (Alexa Fluor® 594), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab45939 staining Vimentin in NIH3T3 cells. The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab45939 at 1μg/ml (shown in green) and ab195889, Mouse monoclonal [DM1A] to alpha Tubulin - Microtubule Marker (Alexa Fluor® 594), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunohistochemical detection (formaldehyde/paraffin-embedded sections) of vimentin protein using vimentin antibody - Neural Stem Cell Marker (ab45939) on human ovarian carcinoma sections. Antigen retrieval step:Heat mediated. Blocking step: 1% BSA for 10 mins at RT°C. Ab45939 was used at 1/700 for 1 hour. Secondary Antibody: Biotin-conjugated anti rabbit IG (1/300).

ab45939 staining Vimentin in murine kidney tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed. Samples were then blocked with 10% serum for 30 minutes at room temperature followed by incubation with the primary antibody at a 1/2100 dilution for 18 hours at 4°C. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/200 dilution.

See Abreview

All lanes : Anti-Vimentin antibody - Cytoskeleton Marker (ab45939) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 3 : HEK293 Human embryonic kidney cell line Whole Cell Lysate
Lane 4 : Ramos (Human Burkitt's lymphoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

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

All lanes : Anti-Vimentin antibody - Cytoskeleton Marker (ab45939) at 1 µg/ml

Lane 1 : Recombinant Human Vimentin protein (ab73843) at 0.1 µg
Lane 2 : Recombinant Human Vimentin protein (ab73843) at 0.01 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

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


Exposure time: 10 seconds


Ab45939 recognizes full length recombinant Human vimentin (ab73843) which has an expected molecular weight of 54 kDa.

Overlay histogram showing MDA-MB-231 cells stained with ab45939 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab45939, 0.01μg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/4000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (polyclonal) (ab171870, 0.01μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Overlay histogram showing NIH3T3 cells stained with ab45939 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab45939, 0.1μg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/4000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (polyclonal) (ab171870, 0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Overlay histogram showing SV40LT-SMC cells stained with ab45939 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab45939, 0.01μg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/4000 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (polyclonal) (ab171870, 0.01μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.