- 700179 - Invitrogen Antibodies CUL2 antibody

Submitted references Unanchored tri-NEDD8 inhibits PARP-1 to protect from oxidative stress-induced cell death.

Keuss MJ, Hjerpe R, Hsia O, Gourlay R, Burchmore R, Trost M, Kurz T
The EMBO journal 2019 Mar 15;38(6)

Differential PROTAC substrate specificity dictated by orientation of recruited E3 ligase.

Smith BE, Wang SL, Jaime-Figueroa S, Harbin A, Wang J, Hamman BD, Crews CM
Nature communications 2019 Jan 10;10(1):131

Characterization of the mammalian family of DCN-type NEDD8 E3 ligases.

Keuss MJ, Thomas Y, Mcarthur R, Wood NT, Knebel A, Kurz T
Journal of cell science 2016 Apr 1;129(7):1441-54

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell, tissue extracts (30 µg lysate) of HEL 92.1.7 (Lane 1), MDA-MB-231 (Lane 2), NTERA-2 (Lane 3), MCF-7 (Lane 4), A549 (Lane 5), Caco-2 (Lane 6), Mouse Testis (Lane 7), Rat Testis (Lane 8) and Mouse pancreas (Lane 9). The blots were probed with Recombinant Rabbit Monoclonal Anti-CUL2 Antibody (Product # 700179, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Recombinant Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A ~ 89 kDa band corresponding to CUL2 was observed across 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 Pierce™ Power Blotter System (Product # 22834). 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: Western blot analysis was performed on whole cell, tissue extracts (30 µg lysate) of HEL 92.1.7 (Lane 1), MDA-MB-231 (Lane 2), NTERA-2 (Lane 3), MCF-7 (Lane 4), A549 (Lane 5), Caco-2 (Lane 6), Mouse Testis (Lane 7), Rat Testis (Lane 8) and Mouse pancreas (Lane 9). The blots were probed with Recombinant Rabbit Monoclonal Anti-CUL2 Antibody (Product # 700179, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Recombinant Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A ~ 89 kDa band corresponding to CUL2 was observed across 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 Pierce™ Power Blotter System (Product # 22834). 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: Knockdown of Cullin 2 was achieved by transfecting NTERA-2 cl.D1 with Cullin 2 specific siRNAs (Silencer® select Product # S16051, S16053). Western blot analysis (Fig. a) was performed using Whole cell extracts from the Cullin 2 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Cullin 2 Recombinant Rabbit Monoclonal Antibody (50H17L12) (Product # 700179 at1 µg/mL concentration) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Cullin 2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Cullin-2 in HeLa cells using a Cullin-2 recombinant rabbit monoclonal antibody (Product # 700179) at a dilution of 10 µg/mL in the absence of peptide (top) and presence of immunogenic peptide (bottom), followed by detection using an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody at a dilution of 1:1000. Actin was stained with Alexa Fluor 568 Phalloidin (Product # A12380). Hoechst only (blue, left), AF488 signal only (green, middle) and composite image with Phalloidin (right).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of CUL-2 was performed using 70% confluent log phase Caco-2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2 % BSA for 1 hour at room temperature. The cells were labeled with CUL-2 (50H17L12) Recombinant Rabbit Monoclonal Antibody (Product # 700179) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Cullin-2 in formalin-fixed, paraffin-embedded human lung (left) and ovarian carcimona (right) using a Cullin-2 monoclonal antibody (Product # 700179) at a dilution of 5 µg/mL. Tissues were pretreated with EDTA and staining was visualized using DAB. Images were taken at a magnification of 20x. Results show strong nuclear staining in tumor cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of Cul-2 was done on K562 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 ABfinity™ Cul-2 Recombinant Rabbit Monoclonal Antibody (700179, red histogram) or with rabbit 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 Goat Anti-Rabbit Secondary Antibody (A11008) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of Cullin-2 in Jurkat cells using a Cullin-2 recombinant rabbit monoclonal antibody (Product # 700179) at a dilution of 0.5 µg. Cells were fixed and permeabilized using FIX & PERM (Product # GAS-004) reagent, and detection was performed using an Alexa Fluor 488 goat anti-rabbit IgG (black) compared to a control without primary antibody (gray). Pre-incubation with the immunogenic peptide decreased the signal (red).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

700179

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