Product nameHuman POR (Cytochrome P450 Reductase) knockout HCT116 cell line
See all Cytochrome P450 Reductase lysates
Parental Cell LineHCT116
Mutation descriptionKnockout achieved by using CRISPR/Cas9, Homozygous: Insertion of the selection cassette in exon 4
Knockout validationSanger Sequencing, Western Blot (WB)
Tested applicationsSuitable for: WBmore details
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.
- 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: 10, 11 D13S317: 10, 12 D7S820: 11, 12 D16S539: 11, 13 vWA: 17, 22 TH01: 8,9 TPOX: 8, 9 CSF1PO: 7, 10
Storage instructionsShipped on Dry Ice. Store in liquid nitrogen.
Storage bufferConstituents: 8.7% DMSO, 2% Cellulose, methyl ether
- Pathways and Processes
- Metabolic signaling pathways
- Lipid and lipoprotein metabolism
FunctionThis 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 diseaseDefects 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 similaritiesIn 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 localizationEndoplasmic reticulum membrane. Anchored to the ER membrane by its N-terminal hydrophobic region.
- Information by UniProt
KO cell lysates
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.
|WB||Use at an assay dependent concentration. Predicted molecular weight: 76 kDa.|
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?
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.
Homozygous: Insertion of the selection cassette in exon4
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.
ab266889 has not yet been referenced specifically in any publications.