Human PDGFRA knockout SH-SY5Y cell line (ab275335)
Overview
-
Product name
Human PDGFRA knockout SH-SY5Y cell line
See all PDGFR alpha lysates -
Parental Cell Line
SHSY-5Y -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 67 bp deletion in exon 3 -
Passage number
<20 -
Knockout validation
Immunocytochemistry (ICC), Sanger Sequencing, Western Blot (WB) -
Tested applications
Suitable for: WB, ICCmore details -
Biosafety level
1 -
General notes
Recommended control: Human wild-type SHSY-5Y cell line (ab275475). 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: 1:1 mixture of EMEM and F-12K + 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 and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
Properties
-
Number of cells
1 x 106 cells/vial, 1 mL -
Viability
~90% -
Tissue
Bone marrow -
Cell type
neuroblastoma -
Disease
Neuroblastoma -
Gender
Female -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
Purity
Immunogen affinity purified -
Research areas
Target
-
Function
Receptor that binds both PDGFA and PDGFB and has a tyrosine-protein kinase activity. -
Tissue specificity
Expressed in primary and metastatic colon tumors and in normal colon tissue. Tumors may express a different isoform to that found in normal tissue. -
Involvement in disease
Note=A chromosomal aberration involving PDGFRA is found in some cases of hypereosinophilic syndrome. Interstitial chromosomal deletion del(4)(q12q12) causes the fusion of FIP1L1 and PDGFRA (FIP1L1-PDGFRA). -
Sequence similarities
Belongs to the protein kinase superfamily. Tyr protein kinase family. CSF-1/PDGF receptor subfamily.
Contains 5 Ig-like C2-type (immunoglobulin-like) domains.
Contains 1 protein kinase domain. -
Cellular localization
Membrane. - Information by UniProt
Associated products
-
KO cell lines
-
Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab275335 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 |
|---|---|---|
| WB |
Use at an assay dependent concentration. Predicted molecular weight: 122 kDa.
|
|
| ICC |
Use at an assay dependent concentration.
|
| Notes |
|---|
|
WB
Use at an assay dependent concentration. Predicted molecular weight: 122 kDa. |
|
ICC
Use at an assay dependent concentration. |
Images
-
Western blot - Human PDGFRA knockout SH-SY5Y cell line (ab275335)All lanes : Anti-PDGFR alpha antibody [EPR22059-270] (ab203491) at 1/1000 dilution
Lane 1 : Wild-type SH-SY5Y cell lysate
Lane 2 : PDGFRA knockout SH-SY5Y cell lysate
Lysates/proteins at 40 µg per lane.
Performed under reducing conditions.
Predicted band size: 122 kDa
Observed band size: 150 kDa why is the actual band size different from the predicted?Lanes 1 - 2: Merged signal (red and green). Green - ab203491 observed at 150 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.
ab203491 was shown to react with PDGFR alpha in wild-type SH-SY5Y cells in Western blot with loss of signal observed in PDGFRA knockout cell line ab275335 (knockout cell lysate ab275522). Wild-type SH-SY5Y and PDGFRA knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab203491 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
-
Immunocytochemistry - Human PDGFRA knockout SH-SY5Y cell line (ab275335)ab203491 staining PDGFR alpha in wild-type SH-SY5Y cells (top panel) and PDGFRA knockout SH-SY5Y cells (bottom panel) (ab275335). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab203491 at 1μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8). -
Western blot - Human PDGFRA knockout SH-SY5Y cell line (ab275335)All lanes : Anti-PDGFR alpha antibody [EPR5480] (ab134123) at 1/1000 dilution
Lane 1 : Wild-type SH-SY5Y cell lysate
Lane 2 : PDGFRA knockout SH-SY5Y cell lysate
Lysates/proteins at 40 µg per lane.
Performed under reducing conditions.
Predicted band size: 122 kDa
Observed band size: 180 kDa why is the actual band size different from the predicted?Lanes 1 - 2: Merged signal (red and green). Green - ab134123 observed at 180 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.
ab134123 was shown to react with PDGFR alpha in wild-type SH-SY5Y cells in Western blot with loss of signal observed in PDGFRA knockout cell line ab275335 (knockout cell lysate ab275522). Wild-type SH-SY5Y and PDGFRA knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab134123 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
-
Sanger Sequencing - Human PDGFRA knockout SH-SY5Y cell line (ab275335)Allele-1: 67 bp deletion in exon 3
Protocols
References (0)
ab275335 has not yet been referenced specifically in any publications.
