Antibodies, Proteins, Kits and Reagents for Life Science

307 566 ₸ Product size

100 µl 307 566 ₸ 10 µl 103 862 ₸

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Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [EPR23221-54] to Eph receptor B4/HTK
Suitable for: WB, IP
Knockout validated
Reacts with: Human

Product name

Anti-Eph receptor B4/HTK antibody [EPR23221-54]
See all Eph receptor B4/HTK primary antibodies
Description

Rabbit monoclonal [EPR23221-54] to Eph receptor B4/HTK
Host species

Rabbit
Tested Applications & Species

Application Species
IP
Human

WB
Human

See all applications and species data
Immunogen

Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
Positive control
- WB: HEK293T, HT-29, HUVEC, HCT 116 and MCF7 lysates. IP: HT-29 and HUVEC whole cell lysates.
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

Application	Species
IP	Human
WB	Human

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Storage buffer

pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR23221-54
Isotype

IgG
Research areas

Western blot - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)

All lanes : Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300) at 1/1000 dilution

Lane 1 : Wild-type HEK293T cell lysate
Lane 2 : EPHB4 knockout HEK293T cell lysate
Lane 3 : MCF7 cell lysate
Lane 4 : HT-29 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 108 kDa
Observed band size: 125 kDa
why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab254300 observed at 125 kDa. Red - loading control ab8245 observed at 36 kDa.

ab254300 Anti-Eph receptor B4/HTK antibody [EPR23221-54] was shown to specifically react with Eph receptor B4/HTK in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266733 (knockout cell lysate ab257429) was used. Wild-type and Eph receptor B4/HTK knockout samples were subjected to SDS-PAGE. ab254300 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
Immunoprecipitation - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)

Eph receptor B4 was immunoprecipitated from 0.35 mg HUVEC (human umbilical vein endothelial cell) whole cell lysate with ab254300 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254300 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1: HUVEC (human umbilical vein endothelial cell) whole cell lysate 10ug

Lane 2: ab254300 IP in HUVEC whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254300 in HUVEC whole cell lysate

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

Exposure time: 8 seconds.

Cleaved intracellular domain (ICD) was observed at around 47kDa. (PMID: 24854540).
Western blot - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)

All lanes : Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300) at 1/1000 dilution

Lane 1 : HT-29 (human colorectal adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : HUVEC (human umbilical vein endothelial cell), whole cell lysate
Lane 3 : SW620 (human colorectal adenocarcinoma epithelial cell), whole cell lysate
Lane 4 : HCT 116 (human colorectal carcinoma epithelial cell), whole cell lysate
Lane 5 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 108 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 37 seconds.

Negative control: SW620 (PMID: 19366806).

EphB4 is negative in SW620 and positive in HT29 and HUVEC, which is different from expression profiles of other EphB family members.

Cleaved intracellular domain (ICD) was observed at around 47kDa. (PMID: 24854540).

The expression profile observed is consistent with what has been described in the literature (PMID: 19366806, 16840724).
Immunoprecipitation - Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)

Eph receptor B4 was immunoprecipitated from 0.35 mg HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate with ab254300 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254300 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1: HT-29 (human colorectal adenocarcinoma epithelial cell) whole cell lysate 10ug

Lane 2: ab254300 IP in HT-29 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254300 in HT-29 whole cell lysate

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

Exposure time: 8 seconds.

Cleaved intracellular domain (ICD) was observed at around 47kDa. (PMID: 24854540).
Anti-Eph receptor B4/HTK antibody [EPR23221-54] (ab254300)

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