Human POR (Cytochrome P450 Reductase) knockout HCT116 cell line (ab266889)
Overview
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Product name
Human POR (Cytochrome P450 Reductase) knockout HCT116 cell line
See all Cytochrome P450 Reductase lysates -
Parental Cell Line
HCT116 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: Insertion of the selection cassette in exon 4 -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: WBmore details -
Biosafety level
1 -
General notes
Recommended control: Human wild-type HCT116 cell line (ab255451). 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: McCoY5a + 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.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 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.
Click here to view the Mammalian cell tissue culture protocol
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.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~90% -
Adherent /Suspension
Adherent -
Tissue
Colon -
Cell type
epithelial -
Disease
Carcinoma -
Gender
Male -
STR Analysis
Amelogenin X D5S818: 10, 11 D13S317: 10, 12 D7S820: 11, 12 D16S539: 11, 13 vWA: 17, 22 TH01: 8,9 TPOX: 8, 9 CSF1PO: 7, 10 -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% DMSO, 2% Cellulose, methyl ether -
Research areas
Target
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Function
This enzyme is required for electron transfer from NADP to cytochrome P450 in microsomes. It can also provide electron transfer to heme oxygenase and cytochrome B5. -
Involvement in disease
Defects in POR are the cause of adrenal hyperplasia variant type (AHV) [MIM:201750]; also known as Antley-Bixler syndrome-like phenotype with disordered steroidogenesis. AHV is a rare variant of congenital adrenal hyperplasia. It is an autosomal recessive disorder with apparent combined P450C17 and P450C21 deficiency. Affected girls are born with ambiguous genitalia, but their circulating androgens are low and virilization does not progress. Conversely, affected boys are sometimes born undermasculinized. Boys and girls can also present with bone malformations, in some cases resembling the pattern seen in patients with Antley-Bixler syndrome.
Defects in POR are a cause of isolated disordered steroidogenesis (IDS) [MIM:201750]. -
Sequence similarities
In the C-terminal section; belongs to the flavoprotein pyridine nucleotide cytochrome reductase family.
Contains 1 FAD-binding FR-type domain.
Contains 1 flavodoxin-like domain. -
Cellular localization
Endoplasmic reticulum membrane. Anchored to the ER membrane by its N-terminal hydrophobic region. - Information by UniProt
Associated products
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KO cell lysates
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Related Products
Applications
Our Abpromise guarantee covers the use of ab266889 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: 76 kDa. |
Images
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All lanes : Anti-Cytochrome P450 Reductase antibody [EPR14479(B)] (ab180597) at 1/1000 dilution
Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : POR knockout HCT116 cell lysate
Lane 3 : HepG2 cell lysate
Lane 4 : A431 cell lysate
Lysates/proteins at 40 µg per lane.
Performed under reducing conditions.
Predicted band size: 76 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?Lanes 1- 4: Merged signal (red and green). Green - ab180597 observed at 80 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab180597 was shown to react with Cytochrome P450 Reductase in wild-type HCT116 cells in western blot. Loss of signal was observed when knockout cell line ab266889 (knockout cell lysate ab257596) was used. Wild-type HCT116 and POR knockout HCT116 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab180597 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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 exon4
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.
References (0)
ab266889 has not yet been referenced specifically in any publications.