Product nameHuman RAD23B (hHR23b) knockout HeLa cell line
See all hHR23b lysates
Parental Cell LineHeLa
Mutation descriptionKnockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon 4
Knockout validationSanger Sequencing, Western Blot (WB)
Tested applicationsSuitable for: WBmore details
Recommended control: Human wild-type HeLa cell line (ab255448). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: DMEM (High Glucose) + 10% FBS
Initial handling guidelines: Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80ºC. Storage at -80ºC may result in loss of viability.
1. Thaw the vial in 37ºC water bath approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 ml) to a 15 ml/50 ml conical sterile polypropylene centrifuge tube containing 8.4 ml pre-warmed culture medium, wash vial with an additional 0.8 ml culture medium (total volume 10 ml) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 ml represents minimum recommended dilution. 20 ml represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 ml pre-warmed culture medium and count using a haemocytometer (Click here to view haemocytometer protocol) or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x104 cells/cm2. This should allow for confluency within 48 hours. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37ºC incubator with 5% CO2. Cultures should be monitored daily.
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended for confluency (80-90% confluence) within 48 hours.
- A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
- Cells should be passaged when they have achieved 80-90% confluence.
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.
Number of cells1 x 106 cells/vial, 1 mL
STR AnalysisAmelogenin 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
Storage instructionsShipped on Dry Ice. Store in liquid nitrogen.
Storage bufferConstituents: 8.7% DMSO, 2% Cellulose, methyl ether
FunctionMultiubiquitin chain receptor involved in modulation of proteasomal degradation. Binds to polyubiquitin chains. Proposed to be capable to bind simultaneously to the 26S proteasome and to polyubiquitinated substrates and to deliver ubiquitinated proteins to the proteasome. May play a role in endoplasmatic reticulum-associated degradation (ERAD) of misfolded glycoproteins by association with PNGase and delivering deglycosylated proteins to the proteasome.
Involved in global genome nucleotide excision repair (GG-NER) by acting as component of the XPC complex. Cooperatively with CETN2 appears to stabilize XPC. May protect XPC from proteasomal degradation.
The XPC complex is proposed to represent the first factor bound at the sites of DNA damage and together with other core recognition factors, XPA, RPA and the TFIIH complex, is part of the pre-incision (or initial recognition) complex. The XPC complex recognizes a wide spectrum of damaged DNA characterized by distortions of the DNA helix such as single-stranded loops, mismatched bubbles or single stranded overhangs. The orientation of XPC complex binding appears to be crucial for inducing a productive NER. XPC complex is proposed to recognize and to interact with unpaired bases on the undamaged DNA strand which is followed by recruitment of the TFIIH complex and subsequent scanning for lesions in the opposite strand in a 5'-to-3' direction by the NER machinery. Cyclobutane pyrimidine dimers (CPDs) which are formed upon UV-induced DNA damage esacpe detection by the XPC complex due to a low degree of structural perurbation. Instead they are detected by the UV-DDB complex which in turn recruits and cooperates with the XPC complex in the respective DNA repair. In vitro, the XPC:RAD23B dimer is sufficient to initiate NER; it preferentially binds to cisplatin and UV-damaged double-stranded DNA and also binds to a variety of chemically and structurally diverse DNA adducts. XPC:RAD23B contacts DNA both 5' and 3' of a cisplatin lesion with a preference for the 5' side. XPC:RAD23B induces a bend in DNA upon binding. XPC:RAD23B stimulates the activity of DNA glycosylases TDG and SMUG1.
Sequence similaritiesBelongs to the RAD23 family.
Contains 1 STI1 domain.
Contains 2 UBA domains.
Contains 1 ubiquitin-like domain.
DomainThe ubiquitin-like domain mediates interaction with ATXN3.
Cellular localizationNucleus. Cytoplasm. The intracellular distribution is cell cycle dependent. Localized to the nucleus and the cytoplasm during G1 phase. Nuclear levels decrease during S-phase; upon entering mitosis, relocalizes in the cytoplasm without association with chromatin.
- Information by UniProt
Our Abpromise guarantee covers the use of ab264961 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use at an assay dependent concentration. Predicted molecular weight: 43 kDa.|
All lanes : Anti-hHR23b antibody [2857D7a] (ab70602) at 1/500 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : RAD23B knockout HeLa cell lysate
Lane 3 : MCF7 cell lysate
Lane 4 : SW620 cell lysate
Lysates/proteins at 20 µg per lane.
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) at 1/10000 dilution
Predicted band size: 43 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?
ab70602 Anti-hHR23b antibody [2857D7a] was shown to specifically react with hHR23b in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264961 (knockout cell lysate ab258623) was used. Wild-type and hHR23b knockout samples were subjected to SDS-PAGE. ab70602 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated at room temperature for 2.5 hours at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
Homozygous: 1 bp insertion in exon 4.
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.
ab264961 has not yet been referenced specifically in any publications.