Human ROBO1 knockout U-87 MG cell line
- Advanced Validation
- What is this?
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- WB
Lab
Western blot - Human ROBO1 knockout U-87 MG cell line (AB306771)
Western blot : Anti-ROBO1 antibody [EPR23699-159] (ab256791) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab256791 was shown to bind specifically to ROBO1. A band was observed at 215 kDa in wild-type U-87 MG cell lysates with no signal observed at this size in ROBO1 knockout cell line. The identity of the lower band is unknown. To generate this image, wild-type and ROBO1 knockout U-87 MG 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes:
Western blot - Anti-Robo1 antibody [EPR23699-159] (<a href='/en-us/products/primary-antibodies/robo1-antibody-epr23699-159-ab256791'>ab256791</a>) at 1/1000 dilution
Lane 1:
Wild-type U-87 MG cell lysate at 20 µg
Lane 2:
ROBO1 knockout U-87 MG cell lysate at 20 µg
Lane 3:
Wild-type HAP1 cell lysate at 20 µg
Lane 4:
ROBO1 knockout HAP1 cell lysate at 20 µg
Secondary
All lanes:
Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Observed band size: 215 kDa
false
- WB
Lab
Western blot - Human ROBO1 knockout U-87 MG cell line (AB306771)
Western blot : Anti-ROBO1 antibody (ab245516) staining at 1/2000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab245516 was shown to bind specifically to ROBO1. A band was observed at 200 kDa in wild-type U-87 MG cell lysates with no signal observed at this size in ROBO1 knockout cell line. To generate this image, wild-type and ROBO1 knockout U-87 MG cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.
All lanes:
Western blot - Anti-Robo1 antibody (<a href='/en-us/products/primary-antibodies/robo1-antibody-ab245516'>ab245516</a>) at 1/2000 dilution
Lane 1:
Wild-type U-87 MG cell lysate at 20 µg
Lane 2:
ROBO1 knockout U-87 MG cell lysate at 20 µg
Secondary
All lanes:
Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution
Observed band size: 200 kDa
false
- NGS
Supplier Data
Next Generation Sequencing - Human ROBO1 knockout U-87 MG cell line (AB306771)
52 bp deletion after Thr97 of the WT protein
Complementary Products
Primary Antibodies
AB256791
Anti-Robo1 antibody [EPR23699-159]
RabMAb|Recombinant|20ul selling size
![Flow Cytometry - Anti-Robo1 antibody [EPR23699-159] (AB256791)](https://content.abcam.com/products/images/robo1-antibody-epr23699-159-ab256791--flow-cytometry-img68058.jpg)
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Reactivity data
Product details
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
- 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
EMEM + 10% FBS
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
Robo1 interacts with Slit proteins to control processes like repulsion of axonal growth cones during neuronal development. The protein acts as an important player in guiding neuronal connections and establishing boundaries. Robo1 does not function alone forming complexes with other receptors and cell adhesion molecules to execute its biological roles effectively. Structurally this receptor facilitates cellular responses to environmental cues through its cytoplasmic signaling cascade.
Pathways
Robo1 engages in the Slit-Robo signaling pathway essential for axonal guidance and cell migration. This pathway involves several key interactions with proteins such as DCC (Deleted in Colorectal Carcinoma) and Netrin impacting neuronal positioning and differentiation. Additionally Robo1 influences cellular orientation and tissue architecture by participating in the actin cytoskeleton remodeling pathway further demonstrating its significance in neuronal patterning.
Cell culture
Biosafety level
EU: 1 US: 1
Adherent/suspension
Adherent
Gender
Male
Target data
Product promise
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