Human SCARB2 knockout MCF7 cell line
- Advanced Validation
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(0 Publication)
- ICC/IF
Collaborator
Immunocytochemistry/ Immunofluorescence - Human SCARB2 knockout MCF7 cell line (AB274952)
ab314217 was shown to react with SCARB2 in wild-type MCF7 cells in immunocytochemistry with loss of signal observed in SCARB2 knockout cell line ab274952. 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 ab314217 at 1/50 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 SCARB2 knockout MCF7 cell line (AB274952)
False colour image of Western blot : Anti-Scavenging Receptor SRB2 antibody [EPR12081] - C-terminal staining at 1/20000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab196651 was shown to bind specifically to Scavenging Receptor SRB2. A band was observed at 80 kDa in wild-type MCF7 cell lysates with no signal observed at this size in SCARB2 knockout cell line ab274952 (knockout cell lysate ab275010). To generate this image, wild-type and SCARB2 knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
All lanes:
Western blot - Anti-Scavenging Receptor SRB2 antibody [EPR12081] - C-terminal (<a href='/en-us/products/primary-antibodies/scavenging-receptor-srb2-antibody-epr12081-c-terminal-ab196651'>ab196651</a>) at 1/20000 dilution
Lane 1:
Wild-type MCF7 cell lysate at 20 µg
Lane 2:
SCARB2 knockout MCF7 cell lysate at 20 µg
Lane 2:
Western blot - Human SCARB2 knockout MCF7 cell line (ab274952)
Predicted band size: 54 kDa
Observed band size: 80 kDa
false
- WB
Lab
Western blot - Human SCARB2 knockout MCF7 cell line (AB274952)
ab196651 was shown to react with SCARB2 in wild-type MCF7 cells in Western blot with loss of signal observed in SCARB2 knockout cell line ab274952. Wild-type MCF7 and SCARB2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab196651 overnight at 4 °C at a 1/20000 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-Scavenging Receptor SRB2 antibody [EPR12081] - C-terminal (<a href='/en-us/products/primary-antibodies/scavenging-receptor-srb2-antibody-epr12081-c-terminal-ab196651'>ab196651</a>) at 1/20000 dilution
Lane 1:
Wild-type MCF7 lysate at 30 µg
Lane 2:
SCARB2 knock-out MCF7 lysate at 30 µg
Lane 2:
Western blot - Human SCARB2 knockout MCF7 cell line (ab274952) at 30 µg
false
- WB
Collaborator
Western blot - Human SCARB2 knockout MCF7 cell line (AB274952)
All lanes:
Western blot - Anti-LIMPII antibody [EPR26243-125] (<a href='/en-us/products/primary-antibodies/limpii-antibody-epr26243-125-ab314217'>ab314217</a>) at 1/1000 dilution
Lane 1:
Wild-type MCF7 lysate at 30 µg
Lane 2:
SCARB2 knock-out MCF7 lysate at 30 µg
Lane 2:
Western blot - Human SCARB2 knockout MCF7 cell line (ab274952) at 30 µg
false
- WB
Collaborator
Western blot - Human SCARB2 knockout MCF7 cell line (AB274952)
ab176317 was shown to react with SCARB2 in wild-type MCF7 cells in Western blot with loss of signal observed in SCARB2 knockout cell line ab274952. Wild-type MCF7 and SCARB2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab176317 overnight at 4 °C at a 1/1000 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-LIMPII antibody [EPR12080] (<a href='/en-us/products/primary-antibodies/limpii-antibody-epr12080-ab176317'>ab176317</a>) at 1/1000 dilution
Lane 1:
Wild-type MCF7 lysate at 30 µg
Lane 2:
SCARB2 knock-out MCF7 lysate at 30 µg
Lane 2:
Western blot - Human SCARB2 knockout MCF7 cell line (ab274952) at 30 µg
false
- WB
Lab
Western blot - Human SCARB2 knockout MCF7 cell line (AB274952)
False colour image of Western blot : Anti-LIMPII antibody [EPR12080] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab176317 was shown to bind specifically to LIMPII. A band was observed at 80 kDa in wild-type MCF7 cell lysates with no signal observed at this size in SCARB2 knockout cell line ab274952 (knockout cell lysate ab275010). To generate this image, wild-type and SCARB2 knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
All lanes:
Western blot - Anti-LIMPII antibody [EPR12080] (<a href='/en-us/products/primary-antibodies/limpii-antibody-epr12080-ab176317'>ab176317</a>) at 1/1000 dilution
Lane 1:
Wild-type MCF7 cell lysate at 20 µg
Lane 2:
Western blot - Human SCARB2 knockout MCF7 cell lysate (<a href='/en-us/products/cell-lysates/human-scarb2-knockout-mcf7-cell-lysate-ab275010'>ab275010</a>) at 20 µg
Lane 3:
SH-SY5Y cell lysate at 20 µg
Lane 4:
HEK-293 cell lysate at 20 µg
Predicted band size: 54 kDa
Observed band size: 80 kDa
false
- NGS
Supplier Data
Next Generation Sequencing - Human SCARB2 knockout MCF7 cell line (AB274952)
1 bp insertion after Leu176 of the WT protein
- NGS
Supplier Data
Next Generation Sequencing - Human SCARB2 knockout MCF7 cell line (AB274952)
Knockout achieved by CRISPR/Cas9; X = 1 bp insertion; Frameshift : 100%
Complementary Products
Primary Antibodies
AB176317
Anti-LIMPII antibody [EPR12080]
RabMAb|Recombinant|KO Validated|20ul selling size
![Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-LIMPII antibody [EPR12080] (AB176317)](https://content.abcam.com/products/images/limpii-antibody-epr12080-ab176317--immunohistochemistry-formalin-pfa-fixed-paraffin-embedded-sections-img134268.jpg)
- 5
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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
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
- Slow to trypsinise.
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 5-7x104 cells/cm2 is recommended.
- Cells should be passaged when they have achieved 80-90% confluence.
Culture medium
MEM + 10% FBS + 0.01 mg/ml bovine insulin
Cryopreservation medium
Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Supplementary information
This supplementary information is collated from multiple sources and compiled automatically.
Biological function summary
The protein plays a critical role in lipid and protein degradation and helps maintain cellular homeostasis. As part of the lysosomal membrane LIMPII facilitates the uptake and processing of large biomolecules into the lysosome. It functions as a receptor for the uptake of β-glucocerebrosidase forming a complex to assist in its proper delivery from the endoplasmic reticulum to the lysosomes. This receptor-ligand relationship ensures efficient enzymatic activity necessary for lipid metabolism.
Pathways
One finds that LIMPII participates in the sphingolipid metabolism pathway and is vital for glucosylceramide catabolism. It interacts with glucocerebrosidase in this pathway which plays a significant role in the metabolism of glycosphingolipids. Another pathway where LIMPII has importance is the autophagy pathway where it contributes to the fusion of autophagosomes with lysosomes. Its activity supports the turnover of cellular components maintaining cellular metabolic balance.
Cell culture
Biosafety level
EU: 1 US: 1
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