Recombinant Anti-ICAM1 antibody [EPR24639-3] KO Tested (ab282575)

Product name

Anti-ICAM1 antibody [EPR24639-3]
See all ICAM1 primary antibodies
Description

Rabbit monoclonal [EPR24639-3] to ICAM1
Host species

Rabbit
Specificity

Rat species is recommended based on Flow Cyt result. We do not guarantee other applications for rat.
Tested applications

Suitable for: Flow Cyt, IP, IHC-P, ICC/IF, WBmore details
Unsuitable for: IHC-Fr
Species reactivity

Reacts with: Rat, Human
Immunogen

Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
Positive control
- WB: Wild-type HeLa whole cell lysate. Ramos and Raji whole cell lysate. Human kidney tissue lysate. IHC-P: Human kidney tissue. Human breast cancer tissue. ICC/IF: Raji cells. Wild-type HeLa cells. Flow Cyt: Rat splenocytes. C6 and Raji cells. IP: Raji whole cell lysate.
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.

Form

Liquid
Storage instructions

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

pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: 59.94% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR24639-3
Isotype

IgG
Research areas
- Neuroscience
- Development

Alternative Versions
- Anti-ICAM1 antibody [EPR24639-3] - BSA and Azide free (ab282599)
Compatible Secondaries
Isotype control
- Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
KO cell lines
- Human ICAM1 knockout HeLa cell line (ab261742)
KO cell lysates
- Human ICAM1 knockout HeLa cell lysate (ab256947)

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab282575 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application	Abreviews	Notes
Flow Cyt		1/500.
IP		1/30.
IHC-P		1/600. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF		1/500.
WB		1/1000. Predicted molecular weight: 57 kDa.

Notes
Flow Cyt 1/500.
IP 1/30.
IHC-P 1/600. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
ICC/IF 1/500.
WB 1/1000. Predicted molecular weight: 57 kDa.

Application notes

Is unsuitable for IHC-Fr.

Function

ICAM proteins are ligands for the leukocyte adhesion protein LFA-1 (integrin alpha-L/beta-2). During leukocyte trans-endothelial migration, ICAM1 engagement promotes the assembly of endothelial apical cups through ARHGEF26/SGEF and RHOG activation. In case of rhinovirus infection acts as a cellular receptor for the virus.
Sequence similarities

Belongs to the immunoglobulin superfamily. ICAM family.
Contains 5 Ig-like C2-type (immunoglobulin-like) domains.
Post-translational
modifications

Monoubiquitinated, which is promoted by MARCH9 and leads to endocytosis.
Cellular localization

Membrane.
Target information above from: UniProt accession P05362 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 3383 Human
- Entrez Gene: 25464 Rat
- Omim: 147840 Human
- SwissProt: P05362 Human
- SwissProt: Q00238 Rat
- Unigene: 643447 Human
- Unigene: 12 Rat
Alternative names
- Antigen identified by monoclonal antibody BB2 antibody
- BB 2 antibody
- BB2 antibody
- CD 54 antibody
- CD_antigen=CD54 antibody
- CD54 antibody
- Cell surface glycoprotein P3.58 antibody
- Human rhinovirus receptor antibody
- ICAM 1 antibody
- ICAM-1 antibody
- ICAM1 antibody
- ICAM1_HUMAN antibody
- intercellular adhesion molecule 1 (CD54) antibody
- intercellular adhesion molecule 1 (CD54), human rhinovirus receptor antibody
- Intercellular adhesion molecule 1 antibody
- Major group rhinovirus receptor antibody
- MALA 2 antibody
- MALA2 antibody
- MyD 10 antibody
- MyD10 antibody
- P3.58 antibody
- Surface antigen of activated B cells antibody
- Surface antigen of activated B cells, BB2 antibody
see all

Western blot - Anti-ICAM1 antibody [EPR24639-3] (ab282575)

All lanes : Anti-ICAM1 antibody [EPR24639-3] (ab282575) at 1/1000 dilution

Lane 1 : Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : ICAM1 knockout HeLa whole cell lysate
Lane 3 : Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at 1/10000 dilution

Predicted band size: 57 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Lanes 1-3: Merged signal (red and green). Green - ab282575 observed at 90kDa. Red - loading control ab8245 observed at 36 kDa.

ab282575 Anti-ICAM1 antibody [EPR24639-3] was shown to react with ICAM1 in wild-type Hela cells in Western blot. Loss of signal was observed when knockout cell line ab261742 (knockout cell lysate ab256947) was used. Wild-type and ICAM1 knockout samples were subjected to SDS-PAGE.

ab282575 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight 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 10000 dilution for 1 hour at room temperature before imaging.
Western blot - Anti-ICAM1 antibody [EPR24639-3] (ab282575)

All lanes : Anti-ICAM1 antibody [EPR24639-3] (ab282575) at 1/1000 dilution

Lane 1 : Wild-type HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : ICAM1 knockout Hela 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: 57 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

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

ab282575 Anti-ICAM1 antibody [EPR24639-3] was shown to react with ICAM1 in wild-type Hela cells in Western blot. Loss of signal was observed when knockout cell line ab261742 (knockout cell lysate ab256947) was used. Wild-type and ICAM1 knockout samples were subjected to SDS-PAGE.

Exposure time: 15 seconds.
Western blot - Anti-ICAM1 antibody [EPR24639-3] (ab282575)

Anti-ICAM1 antibody [EPR24639-3] (ab282575) at 1/1000 dilution + human kidney tissue lysate at 20 µg

Secondary
VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 57 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

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

Exposure time: 26 seconds
Western blot - Anti-ICAM1 antibody [EPR24639-3] (ab282575)

All lanes : Anti-ICAM1 antibody [EPR24639-3] (ab282575) at 1/1000 dilution

Lane 1 : Ramos (human Burkitt's lymphoma B lymphocyte) whole cell lysate
Lane 2 : Raji (human Burkitts lymphoma B lymphocyte) 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: 57 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

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

Exposure time: 15 seconds
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ICAM1 antibody [EPR24639-3] (ab282575)

Immunohistochemical analysis of paraffin-embedded human kidney tissue labelling ICAM1 with ab282575 at 1/600 (0.902 µg/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on normal human glomerulus. The section was incubated with ab282575 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ICAM1 antibody [EPR24639-3] (ab282575)

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labelling ICAM1 with ab282575 at 1/600 (0.902 µg/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human breast cancer (PMID: 30082828). The section was incubated with ab282575 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
Immunocytochemistry/ Immunofluorescence - Anti-ICAM1 antibody [EPR24639-3] (ab282575)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Raji cells labelling ICAM1 with ab282575 at 1/500 (1.082 µg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in Raji cells.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.
Immunocytochemistry/ Immunofluorescence - Anti-ICAM1 antibody [EPR24639-3] (ab282575)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized ICAM1 KO HeLa cells labelling ICAM1 with ab282575 at 1/500 (1.082 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in wild-type HeLa cells, and no staining in ICAM1 knockout HeLa cells is observed.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 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 dilution.
Flow Cytometry - Anti-ICAM1 antibody [EPR24639-3] (ab282575)

Flow cytometric analysis of rat splenocytes cells labelling ICAM1 with ab282575 at 1/500 dilution (0.1 µg)/ Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. A goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.
Flow Cytometry - Anti-ICAM1 antibody [EPR24639-3] (ab282575)

Flow cytometric analysis of C6 (Rat glial tumor glial cell) cells labelling ICAM1 with ab282575 at 1/500 dilution (0.1 µg)/(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, ab150077) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.
Flow Cytometry - Anti-ICAM1 antibody [EPR24639-3] (ab282575)

Flow cytometric analysis of Raji (Human Burkitt's lymphoma B lymphocyte) cells labelling ICAM1 with ab282575 at 1/500 dilution (0.1 µg)/(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, ab150077) at 1/2000 dilution was used as the secondary antibody.

Gated on viable cells.
Immunoprecipitation - Anti-ICAM1 antibody [EPR24639-3] (ab282575)

ICAM1 was immunoprecipitated from 0.35 mg Raji (human burkitt's lymphoma b lymphocyte) whole cell lysate 10 µg with ab282575 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab282575 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Raji whole cell lysate 10 µg

Lane 2: ab282575 IP in Raji whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab282575 in Raji whole cell lysate

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

Exposure time: 3 minutes.
Anti-ICAM1 antibody [EPR24639-3] (ab282575)

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

SDS download

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Key features and details

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Overview

Product name

Description

Host species

Specificity

Tested applications

Species reactivity

Immunogen

Positive control

General notes

Properties

Form

Storage instructions

Storage buffer

Purity

Clonality

Clone number

Isotype

Research areas

Associated products

Alternative Versions

Compatible Secondaries

Isotype control

KO cell lines

KO cell lysates

Applications

The Abpromise guarantee

Target

Function

Sequence similarities

Post-translationalmodifications

Cellular localization

Database links

Alternative names

Images

Protocols

Datasheets and documents

SDS download

Datasheet download

Certificate of Compliance

References (0)

Customer reviews and Q&As

Post-translational
modifications