Human PARK7 (DJ1) knockout HEK-293T cell line
- Advanced Validation
- What is this?
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(0 Publication)
- ICC/IF
Collaborator
Immunocytochemistry/ Immunofluorescence - Human PARK7 (DJ1) knockout HEK-293T cell line (AB266338)
ab201147 was shown to react with PARK7 in wild-type HEK-293T cells in immunocytochemistry with loss of signal observed in PARK7 knockout cell line ab266338. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab201147 at 1/500 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 µg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).
This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.
- ICC/IF
Collaborator
Immunocytochemistry/ Immunofluorescence - Human PARK7 (DJ1) knockout HEK-293T cell line (AB266338)
- ICC/IF
Collaborator
Immunocytochemistry/ Immunofluorescence - Human PARK7 (DJ1) knockout HEK-293T cell line (AB266338)
ab76241 was shown to react with PARK7 in wild-type HEK-293T cells in immunocytochemistry with loss of signal observed in PARK7 knockout cell line ab266338. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab76241 at 1/100 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 µg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).
This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.
- WB
Lab
Western blot - Human PARK7 (DJ1) knockout HEK-293T cell line (AB266338)
Lanes 1-4 : Merged signal (red and green). Green - ab76008 observed at 24 kDa. Red - loading control ab8245 observed at 36 kDa.
ab76008 Anti-PARK7/DJ1 antibody [EP2815Y] was shown to specifically react with PARK7/DJ1 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266338 (knockout cell lysate ab257016) was used. Wild-type and PARK7/DJ1 knockout samples were subjected to SDS-PAGE. ab76008 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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.
All lanes:
Western blot - Anti-PARK7/DJ1 antibody [EP2815Y] (<a href='/en-us/products/primary-antibodies/park7-dj1-antibody-ep2815y-ab76008'>ab76008</a>) at 1/1000 dilution
Lane 1:
Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 20 µg
Lane 2:
PARK7 knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate at 20 µg
Lane 2:
Western blot - Human PARK7 (DJ1) knockout HEK-293T cell line (ab266338)
Lane 3:
HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 4:
Human brain nuclear fraction tissue lysate at 20 µg
Secondary
All lanes:
Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution
Predicted band size: 20 kDa
Observed band size: 24 kDa
false
- WB
Collaborator
Western blot - Human PARK7 (DJ1) knockout HEK-293T cell line (AB266338)
ab76008 was shown to react with PARK7 in wild-type HEK-293T cells in Western blot with loss of signal observed in PARK7 knockout cell line ab266338. Wild-type HEK-293T and PARK7 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab76008 overnight at 4 °C at a 1/5000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.
This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.
All lanes:
Western blot - Anti-PARK7/DJ1 antibody [EP2815Y] (<a href='/en-us/products/primary-antibodies/park7-dj1-antibody-ep2815y-ab76008'>ab76008</a>) at 1/5000 dilution
Lane 1:
Wild-type HEK-293T lysate at 30 µg
Lane 2:
PARK7 knock-out HEK-293T lysate at 30 µg
Lane 2:
Western blot - Human PARK7 (DJ1) knockout HEK-293T cell line (ab266338) at 30 µg
false
- Cell Culture
Lab
Cell Culture - Human PARK7 (DJ1) knockout HEK-293T cell line (AB266338)
Representative images PARK7 knockout HEK293T cells, low and high confluency examples (top left and right respectively) and wild-type HEK293T cells, low and high confluency (bottom left and right respectively) showing typical adherent, epithelial-like morphology. Images were captured at 10X magnification using a EVOS M5000 microscope.
- Sanger seq
Unknown
Sanger Sequencing - Human PARK7 (DJ1) knockout HEK-293T cell line (AB266338)
Homozygous : Insertion of the selection cassette in exon 2
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Reactivity data
Product details
We will provide viable cells that proliferate on revival.
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.
What's included?
Properties and storage information
Gene name
Gene editing type
Gene editing method
Knockout validation
Zygosity
Shipped at conditions
Appropriate short-term storage conditions
Appropriate long-term storage conditions
Handling procedures
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 seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate 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. 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.
- Cells should be passaged when they have achieved 80-90% confluence.
Culture medium
DMEM (High Glucose) + 10% FBS
Cryopreservation medium
Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Quality control
STR analysis
CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX
Cell culture
Biosafety level
EU: 2 US: 2
Adherent/suspension
Adherent
Gender
Female
Target data
Product promise
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
For licensing inquiries, please contact partnerships@abcam.com