Human ICAM1 knockout HeLa cell lysate (ab256947)
Overview
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Product name
Human ICAM1 knockout HeLa cell lysate
See all ICAM1 kits -
Product overview
Knockout cell lysate achieved by CRISPR/Cas9. -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp insertion in exon 2 and Insertion of the selection cassette in exon 2. -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Reconstitution notes
To use as WB control, resuspend the lyophilizate in 50 µL of LDS* Sample Buffer to have a final concentration of 2 mg/ml. For reducing conditions, we recommend a final concentration of 0.1 M DTT.*Usage of SDS sample buffer is not recommended with these lyophilized lysates.
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Notes
Lysate preparation: Our lysates are made using RIPA buffer to which we add a protease inhibitor cocktail and phosphatase inhibitor cocktail (ratio: 300:100:10). This means that the protein of interest is denatured. If you require a native form of the protein please use the live cell version - found here. Please refer to our lysis protocol for further details on how our lysates are prepared.
User storage instructions: Lyophilizate may be stored at 4°C. After reconstitution, store at -20°C for short-term storage or -80°C for long-term storage.
Access thousands of knockout cell lysates, generated from commonly used cancer cell lines.
See here for more information on knockout cell lysates.Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.
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Tested applications
Suitable for: WBmore details
Properties
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Storage instructions
Store at -80°C. Please refer to protocols. -
Components 1 kit ab261925 - Human ICAM1 knockout HeLa cell lysate 1 x 100µg ab255552 - Human wild-type HeLa cell lysate 1 x 100µg -
Research areas
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Cell type
epithelial -
Disease
Adenocarcinoma -
Gender
Female -
STR Analysis
Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8, 12 CSF1PO: 9, 10
Target
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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
modificationsMonoubiquitinated, which is promoted by MARCH9 and leads to endocytosis. -
Cellular localization
Membrane. - Information by UniProt
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Alternative names
- Antigen identified by monoclonal antibody BB2
- BB 2
- BB2
see all
Associated products
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KO cell lines
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab256947 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 |
|---|---|---|
| WB |
Use at an assay dependent concentration. Predicted molecular weight: 57 kDa.
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| Notes |
|---|
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WB
Use at an assay dependent concentration. Predicted molecular weight: 57 kDa. |
Images
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Western blot - Human ICAM1 knockout HeLa cell lysate (ab256947)
Lane 1: Wild-type HeLa cell lysate 20 μg
Lane 2: ICAM1 knockout HeLa cell lysate 20 μg
Lane 3: A549 cell lysate 20 μg
Lane 4: Ramos cell lysate 20 μg
False colour image of Western blot: Anti-ICAM1 antibody [EP1442Y] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab53013 was shown to bind specifically to ICAM1. A band was observed at 90 kDa (not observed by this antibody) in wild-type HeLa cell lysates with no signal observed at this size in Icam1 knockout cell line ab261742 (knockout cell lysate ab256947). The band observed in the knockout lysate lane below 90 kDa is likely to represent a truncated form of ICAM1. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and Icam1 knockout HeLa cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution. -
Western blot - Human ICAM1 knockout HeLa cell lysate (ab256947)Lane 1: Wild-type HeLa cell lysate 20 μg
Lane 2: ICAM1 knockout HeLa cell lysate 20 μg
Lane 3: A549 cell lysate 20 μg
Lane 4: Ramos cell lysate 20 μg
False colour image of Western blot: Anti-ICAM1 antibody [EPR4776] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109361 was shown to bind specifically to ICAM1. A band was observed at 90 kDa in wild-type HeLa cell lysates with no signal observed at this size in Icam1 knockout cell line ab261742 (knockout cell lysate ab256947). The band observed in the knockout lysate lane below 90 kDa is likely to represent a truncated form of ICAM1. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and Icam1 knockout HeLa cell lysates were analysed.First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution. -
Western blot - Human ICAM1 knockout HeLa cell lysateBlocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS
Lane 1: Wild-type HeLa cell lysate
Lane 2: ICAM1 knockout HeLa cell lysate
Lane 3: Ramos cell lysateLanes 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 4oC 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.
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Western blot - Human ICAM1 knockout HeLa cell lysate (ab256947)Lane 1:Wild-type HeLa cell lysate (20 ug)
Lane 2:ICAM1 knockout HeLa cell lysate (20 ug)
Lane 3:Raji cell lysate (20 ug)
Lane 4:Ramos cell lysate (20 ug)ab53013 was shown to specifically react with ICAM1 in wild-type HeLa cells. 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. ab53013 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4oC 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.
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Sanger Sequencing - Human ICAM1 knockout HeLa cell lysate (ab256947)Allele-1: Insertion of the selection cassette in exon 2
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Sanger Sequencing - Human ICAM1 knockout HeLa cell lysate (ab256947)Allele-2: 1 bp insertion in exon 2
Protocols
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.
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab256947 has not yet been referenced specifically in any publications.