Human SP1 knockout HeLa cell line (ab265519)
Overview
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Product name
Human SP1 knockout HeLa cell line
See all SP1 lysates -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: Insertion of the selection cassette in exon 2 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: WBmore details -
Biosafety level
2 -
General notes
Recommended control: Human wild-type HeLa cell line (ab255928). 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 -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for 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 or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37°C incubator with 5% CO2. Cultures should be monitored daily.Subculture guidelines:
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended.
- 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.
We will provide viable cells that proliferate on revival.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Cervix -
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 -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
Research areas
Target
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Function
Transcription factor that can activate or repress transcription in response to physiological and pathological stimuli. Binds with high affinity to GC-rich motifs and regulates the expression of a large number of genes involved in a variety of processes such as cell growth, apoptosis, differentiation and immune responses. Highly regulated by post-translational modifications (phosphorylations, sumoylation, proteolytic cleavage, glycosylation and acetylation). Binds also the PDGFR-alpha G-box promoter. May have a role in modulating the cellular response to DNA damage. Implicated in chromatin remodeling. Plays a role in the recruitment of SMARCA4/BRG1 on the c-FOS promoter. Plays an essential role in the regulation of FE65 gene expression. In complex with ATF7IP, maintains telomerase activity in cancer cells by inducing TERT and TERC gene expression. -
Tissue specificity
Up-regulated in adenocarcinomas of the stomach (at protein level). -
Sequence similarities
Belongs to the Sp1 C2H2-type zinc-finger protein family.
Contains 3 C2H2-type zinc fingers. -
Post-translational
modificationsPhosphorylated on multiple serine and threonine residues. Phosphorylation is coupled to ubiquitination, sumoylation and proteolytic processing. Phosphorylation on Ser-59 enhances proteolytic cleavage. Phosphorylation on Ser-7 enhances ubiquitination and protein degradation. Hyperphosphorylation on Ser-101 in response to DNA damage has no effect on transcriptional activity. MAPK1/MAPK3-mediated phosphorylation on Thr-453 and Thr-739 enhances VEGF transcription but, represses FGF2-triggered PDGFR-alpha transcription. Also implicated in the repression of RECK by ERBB2. Hyperphosphorylated on Thr-278 and Thr-739 during mitosis by MAPK8 shielding SP1 from degradation by the ubiquitin-dependent pathway. Phosphorylated in the zinc-finger domain by calmodulin-activated PKCzeta. Phosphorylation on Ser-641 by PKCzeta is critical for TSA-activated LHR gene expression through release of its repressor, p107. Phosphorylation on Thr-668, Ser-670 and Thr-681 is stimulated by angiotensin II via the AT1 receptor inducing increased binding to the PDGF-D promoter. This phosphorylation is increased in injured artey wall. Ser-59 and Thr-681 can both be dephosphorylated by PP2A during cell-cycle interphase. Dephosphorylation on Ser-59 leads to increased chromatin association during interphase and increases the transcriptional activity. On insulin stimulation, sequentially glycosylated and phosphorylated on several C-terminal serine and threonine residues.
Acetylated. Acetylation/deacetylation events affect transcriptional activity. Deacetylation leads to an increase in the expression the 12(s)-lipooxygenase gene though recruitment of p300 to the promoter.
Ubiquitinated. Ubiquitination occurs on the C-terminal proteolytically-cleaved peptide and is triggered by phosphorylation.
Sumoylated by SUMO1. Sumoylation modulates proteolytic cleavage of the N-terminal repressor domain. Sumoylation levels are attenuated during tumorigenesis. Phosphorylation mediates SP1 desumoylation.
Proteolytic cleavage in the N-terminal repressor domain is prevented by sumoylation. The C-terminal cleaved product is susceptible to degradation.
O-glycosylated; contains at least 8 N-acetylglucosamine side chains. Levels are controlled by insulin and the SP1 phosphorylation states. Insulin-mediated O-glycosylation locates SP1 to the nucleus, where it is sequentially deglycosylated and phosphorylated. O-glycosylation affects transcriptional activity through disrupting the interaction with a number of transcription factors including ELF1 and NFYA. Also inhibits interaction with the HIV1 promoter. Inhibited by peroxisomome proliferator receptor gamma (PPARgamma). -
Cellular localization
Nucleus. Cytoplasm. Nuclear location is governed by glycosylated/phosphorylated states. Insulin promotes nuclear location, while glucagon favors cytoplasmic location. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab265519 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. Predicted molecular weight: 81 kDa.
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Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 81 kDa. |
Images
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All lanes : Anti-SP1 antibody [EPR22648-50] - ChIP Grade (ab231778) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : SP1 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?Lanes 1-2: Merged signal (red and green). Green - ab231778 observed at 100 kDa. Red - loading control ab8245 observed at 37 kDa.
ab231778 Anti-SP1 antibody [EPR22648-50] was shown to specifically react with SP1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265519 (knockout cell lysate ab257698) was used. Wild-type and SP1 knockout samples were subjected to SDS-PAGE. ab231778 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 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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All lanes : Anti-SP1 antibody [EPR6662(B)] (ab124804) at 1/5000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : SP1 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 81 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?Lanes 1-2: Merged signal (red and green). Green - ab124804 observed at 100 kDa. Red - loading control ab8245 observed at 37 kDa.
ab124804 Anti-SP1 antibody [EPR6662(B)] was shown to specifically react with SP1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265519 (knockout cell lysate ab257698) was used. Wild-type and SP1 knockout samples were subjected to SDS-PAGE. ab124804 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 5000 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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Homozygous: Insertion of the selection cassette in exon 2.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab265519 has not yet been referenced specifically in any publications.