Anti-Vimentin (phospho S56) 抗体 [EPR21084]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Vimentin (phospho S56) antibody [EPR21084] (AB217673)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Vimentin (phospho S56) with ab217673 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing strong positive staining in HeLa cells in M phase.

The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control : PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Vimentin (phospho S56) antibody [EPR21084] (AB217673)

ab217673 staining Vimentin (phospho S56) in HeLa (human cervix adenocarcinoma epithelial cell) cells by intracellular flow cytometry. Cells were fixed using 80% Methanol and permeabilized with 0.1% Tween-20. The sample was incubated with primary antibody at 1/500 dilution. An Alexa Fluor^®488 Goat anti-rabbit IgG (ab150077) was used as a secondary antibody. Rabbit monoclonal IgG (ab172730) was used as an isotype control (left). Cells were pre-treated with 20 μg/ml RNase A for 30 minutes to eliminate the non-specific binding between RNA and PI (Propidium iodide). Vimentiin (phospho S56) is highly expressed in mitotic cells. (PMID : 16260496)

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Vimentin (phospho S56) antibody [EPR21084] (AB217673)

Flow cytometry overlay histogram showing HeLa treated with 100ng/mL Nocodazole for 18 hours + RNAse (green line) and untreated HeLa (magenta line) stained with ab217673. The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Tween 20 for 20 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab217673) (1x 10⁶in 100μl at 0.04μg/ml (1/56000)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

IIsotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (treated HeLa - black line, untreated HeLa - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity)

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IP

Supplier Data

Immunoprecipitation - Anti-Vimentin (phospho S56) antibody [EPR21084] (AB217673)

Vimentin (phospho S56) was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) treated with 100 ng/ml nocodazole (ab120630) for 18 hours, whole cell lysate with ab217673 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab217673 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa treated with 100 ng/ml nocodazole (ab120630) for 18 hours, whole cell lysate 10 μg (Input).
Lane 2 : ab217673 IP in HeLa treated with 100 ng/ml nocodazole (ab120630) for 18 hours, whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab217673 in HeLa treated with 100 ng/ml nocodazole (ab120630) for 18 hours, whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Vimentin (phospho S56) antibody [EPR21084] (ab217673)

Predicted band size: 53 kDa

Observed band size: 57 kDa

true

Exposure time: 1s

• WB

Lab

Western blot - Anti-Vimentin (phospho S56) antibody [EPR21084] (AB217673)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Vimentin (phospho S56) antibody [EPR21084] (ab217673) at 1/1000 dilution

Lane 1:

Untreated HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

HeLa (human epithelial cell line from cervix adenocarcinoma) treated with 100 ng/ml nocodazole (<a href='/products/biochemicals/nocodazole-microtubule-inhibitor-ab120630'>ab120630</a>) for 18 hours, whole cell lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 53 kDa

Observed band size: 57 kDa

true

Exposure time: 23s

• Dot

Supplier Data

Dot Blot - Anti-Vimentin (phospho S56) antibody [EPR21084] (AB217673)

Dot blot analysis of Vimentin (phospho S56) labeled with ab217673 at 1/1000 dilution.

Lane 1 : Vimentin (phospho S56) peptide.
Lane 2 : Vimentin non-phospho peptide.

Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure time : 1 minute.

The blot was developed on a BIO-RAD® ChemiDoc™ MP instrument.