Human CDK4 knockout HeLa cell lysate (ab263780)
Overview
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Product name
Human CDK4 knockout HeLa cell lysate -
Product overview
Knockout cell lysate achieved by CRISPR/Cas9. -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 139 bp insertion in exon2 and 2 bp deletion in exon2. -
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 ab255482 - Human CDK4 knockout HeLa cell lysate 1 x 100µg ab255929 - 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
Ser/Thr-kinase component of cyclin D-CDK4 (DC) complexes that phosphorylate and inhibit members of the retinoblastoma (RB) protein family including RB1 and regulate the cell-cycle during G(1)/S transition. Phosphorylation of RB1 allows dissociation of the transcription factor E2F from the RB/E2F complexes and the subsequent transcription of E2F target genes which are responsible for the progression through the G(1) phase. Hypophosphorylates RB1 in early G(1) phase. Cyclin D-CDK4 complexes are major integrators of various mitogenenic and antimitogenic signals. Also phosphorylates SMAD3 in a cell-cycle-dependent manner and represses its transcriptional activity. Component of the ternary complex, cyclin D/CDK4/CDKN1B, required for nuclear translocation and activity of the cyclin D-CDK4 complex. -
Involvement in disease
Defects in CDK4 are a cause of susceptibility to cutaneous malignant melanoma type 3 (CMM3) [MIM:609048]. Malignant melanoma is a malignant neoplasm of melanocytes, arising de novo or from a pre-existing benign nevus, which occurs most often in the skin but also may involve other sites. -
Sequence similarities
Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. CDC2/CDKX subfamily.
Contains 1 protein kinase domain. -
Post-translational
modificationsPhosphorylation at Thr-172 is required for enzymatic activity. Phosphorylated, in vitro, at this site by CCNH-CDK7, but, in vivo, appears to be phosphorylated by a proline-directed kinase. In the cyclin D-CDK4-CDKN1B complex, this phosphorylation and consequent CDK4 enzyme activity, is dependent on the tyrosine phosphorylation state of CDKN1B. Thus, in proliferating cells, CDK4 within the complex is phosphorylated on Thr-172 in the T-loop. In resting cells, phosphorylation on Thr-172 is prevented by the non-tyrosine-phosphorylated form of CDKN1B. -
Cellular localization
Cytoplasm. Nucleus. Membrane. Cytoplasmic when non-complexed. Forms a cyclin D-CDK4 complex in the cytoplasm as cells progress through G(1) phase. The complex accumulates on the nuclear membrane and enters the nucleus on transition from G(1) to S phase. Also present in nucleoli and heterochromatin lumps. Colocalizes with RB1 after release into the nucleus. - Information by UniProt
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Alternative names
- Cdk 4
- cdk4
- CDK4 protein
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 ab263780 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 |
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WB |
Use at an assay dependent concentration.
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Notes |
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WB
Use at an assay dependent concentration. |
Images
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Lane 1: Wild-type HeLa cell lysate (20µg)
Lane 2: CDK4 knockout HeLa cell lysate (20µg)
Lanes 1- 2: Merged signal (red and green). Green - ab199728 observed at 34 kDa. Red - loading control ab130007 observed at 124 kDa.
ab199728 Recombinant Anti-Cdk4 antibody [EPR17525] was shown to specifically react with CDK4 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255378 (knockout cell lysate ab263780) was used. Wild-type and CDK4 knockout samples were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab199728 and Anti-Vinculin antibody [VIN-54] were incubated overnight at 4°C at 1 in 2000 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. -
Lane 1: Wild-type HeLa cell lysate (20µg)
Lane 2: CDK4 knockout HeLa cell lysate (20µg)
Lanes 1- 2: Merged signal (red and green). Green - ab108357 observed at 34 kDa. Red - loading control ab130007 observed at 124 kDa.
ab108357 Recombinant Anti-Cdk4 antibody [EPR4513-32-7] was shown to specifically react with CDK4 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255378 (knockout cell lysate ab263780) was used. Wild-type and CDK4 knockout samples were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab108357 and Anti-Vinculin antibody [VIN-54] 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. -
Allele-1: 2 bp deletion in exon2; Allele-2: 139 bp insertion in exon2
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)
ab263780 has not yet been referenced specifically in any publications.