Anti-EPCR/CD201 antibody [EPR25647-5]

• Flow Cyt

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Flow Cytometry - Anti-EPCR/CD201 antibody [EPR25647-5] (AB300525)

Flow cytometric analysis of Jurkat (human T cell leukemia T lymphocyte, Left) / HUVEC (human umbilical vein endothelial cell, Right) cells labelling EPCR/CD201 with ab300525 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Gated on viable cells.Negative control : Jurkat (PMID : 11703343).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-EPCR/CD201 antibody [EPR25647-5] (AB300525)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HUVEC (human umbilical vein endothelial cell) cells labelling EPCR/CD201 with ab300525 at 1/50 (9.8 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing cytoplasmic and membranous staining in HUVEC cell lineNegative control : Jurkat (PMID : 11703343) is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• IP

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Immunoprecipitation - Anti-EPCR/CD201 antibody [EPR25647-5] (AB300525)

EPCR/CD201 was immunoprecipitated from 0.35 mg HUVEC (human umbilical vein endothelial cell) whole cell lysate 10 ug with ab300525 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300525 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP)(ab131366) was used at 1/1000 dilution. Lane 1 : HUVEC (human umbilical vein endothelial cell) whole cell lysate 10 ug Lane 2 : ab300525 IP in HUVEC whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab300525 in HUVEC whole cell lysate. Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 3 minutes

Lane 1:

Immunoprecipitation - Anti-EPCR/CD201 antibody [EPR25647-5] (ab300525) at 1/30 dilution

Lane 2:

Immunoprecipitation - Anti-EPCR/CD201 antibody [EPR25647-5] (ab300525) at 1/1000 dilution

All lanes:

HUVEC whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 3min

• WB

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Western blot - Anti-EPCR/CD201 antibody [EPR25647-5] (AB300525)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Negative control : Jurkat (PMID : 7929370)

Exposure time : Lanes 1-4 : 103 seconds; Lanes 5-6 : 3 minutes

All lanes:

Western blot - Anti-EPCR/CD201 antibody [EPR25647-5] (ab300525) at 1/1000 dilution

Lane 1:

HUVEC (human umbilical vein endothelial cell), whole cell lysate at 10 µg

Lane 2:

Ea.Hy926 (human somatic cell hybrid endothelial), whole cell lysate at 10 µg

Lane 3:

RKO (human colon epithelial cell), whole cell lysate at 10 µg

Lane 4:

Jurkat (human T cell leukemia T lymphocyte), whole cell lysate at 10 µg

Lane 5:

Human placenta tissue lysate at 10 µg

Lane 6:

Human lung tissue 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 1/20000 dilution

Predicted band size: 27 kDa

Observed band size: 45 kDa

false

• WB

Lab

Western blot - Anti-EPCR/CD201 antibody [EPR25647-5] (AB300525)

Exposure time : ab300525 : 180 seconds; ab300565 : 5 seconds

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

The band beneath the target band is supposed to be a soluble form (PMID : 19806250).

ab300525 is less sensitive than ab300565. ab300525 needs a high sensitivity ECL substrate.

Lanes 1-5 & 11-15 use a normal normal ECL substrate.
Lanes 6-10 use a High sensitivity ECL substrate.

Lanes 1 - 15:

Western blot - Anti-EPCR/CD201 antibody [EPR25647-5] (ab300525) at 1/1000 dilution

Lanes 11 - 15:

Western blot - Anti-EPCR/CD201 antibody [EPR25647-49] (<a href='/en-us/products/primary-antibodies/epcr-cd201-antibody-epr25647-49-ab300565'>ab300565</a>) at 1/1000 dilution

Lanes 1, 6 and 11:

Human lung tissue lysate at 20 µg

Lanes 2, 7 and 12:

HUVEC (Human umbilical vein endothelial cell) whole cell lysate at 20 µg

Lanes 3, 8 and 13:

Ea.Hy926 ( human somatic cell hybrid endothelial) whole cell lysate at 20 µg

Lanes 4, 9 and 14:

RKO (human colorectal carcinoma cell) whole cell lysate at 20 µg

Lanes 5, 10 and 15:

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

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 45 kDa

false

• WB

Supplier Data

Western blot - Anti-EPCR/CD201 antibody [EPR25647-5] (AB300525)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-EPCR/CD201 antibody [EPR25647-5] (ab300525) at 1/1000 dilution

Lane 1:

Untreated HUVEC whole cell lysate at 10 µg

Lane 2:

HUVEC whole cell lysate treated with Protein Deglycosylation Mix II 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 1/20000 dilution

Predicted band size: 27 kDa

Observed band size: 45 kDa

true

Exposure time: 3min