- MA5-12795 - Invitrogen Antibodies PTPRC antibody

Shin JU, Kim SH, Kim H, Noh JY, Jin S, Park CO, Lee WJ, Lee DW, Lee JH, Lee KH
The Journal of investigative dermatology 2016 Feb;136(2):507-515

Deng G, Krishnakumar S, Powell AA, Zhang H, Mindrinos MN, Telli ML, Davis RW, Jeffrey SS
BMC cancer 2014 Jun 19;14:456

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of CD45 was performed using membrane enriched extracts (30 µg lysate) of KARPAS 299 (Lane 1) and THP-1 (Lane 2). The blots were probed with Anti-CD45 Mouse Monoclonal Antibody (Product # MA5-12795, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). A 220 kDa band corresponding to CD45 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with overnight wet transfer system. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of CD45 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRIPSR664208_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of CD45 was performed by loading 30 µg of THP-1 Cas9 (Lane 1) andTHP-1 CD45 KO (Lane 2) membrane enriched extracts. The samples were electrophoresed using NuPAGE™ 3 to 8%, Tris-Acetate, 1.0 mm, Mini Protein Gel (Product # EA0375PK2). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with a CD45 Monoclonal Antibody (Bra55) (Product # MA5-12795, 1:100 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:2,500 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to CD45. An uncharacterised band was observed at ~60 kDa in THP-1 Cas9 sample.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of CD45/PTPRC was performed using Jurkat cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with CD45/PTPRC Mouse Monoclonal Antibody (MA5-12795, red histogram) or with mouse isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Rabbit Anti-Mouse Secondary Antibody (A11059) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control..

MA5-12795