- PA1-752 - Invitrogen Antibodies APBB1 antibody

Submitted references Age-related accumulation of Reelin in amyloid-like deposits.

Knuesel I, Nyffeler M, Mormède C, Muhia M, Meyer U, Pietropaolo S, Yee BK, Pryce CR, LaFerla FM, Marighetto A, Feldon J
Neurobiology of aging 2009 May;30(5):697-716

RNA interference silencing of the adaptor molecules ShcC and Fe65 differentially affect amyloid precursor protein processing and Abeta generation.

Xie Z, Dong Y, Maeda U, Xia W, Tanzi RE
The Journal of biological chemistry 2007 Feb 16;282(7):4318-4325

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed using tissue and membrane enriched extracts (30 µg lysate) of U-87 MG (Lane 1), SH-SY5Y (Lane 2), NTERA-2 (Lane 3), U-2 OS (Lane 4), A549 (Lane 5), Mouse Brain (Lane 6) and PC-12 (Lane 7). The blots were probed with Anti-Fe65 Rabbit Polyclonal Antibody (Product # PA1-752, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A ~ 77 kDa band corresponding to Fe65 was observed across cell lines and tissue tested. Additionally ~ 41 kDa band was observed in U-87 MG, A549, and Mouse brain. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), 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 iBlot® 2 Dry Blotting System (Product # IB21001). 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 of Fe65-affinity pulldown materials from mouse brain lysate using Product # PA1-752. Lane 1 is an immunopurified sample from a full-length Fe65 Knockout mouse. Lane 2 is an immunopurified sample from an Fe65 wild type mouse.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed using tissue and membrane enriched extracts (30 µg lysate) of U-87 MG (Lane 1), SH-SY5Y (Lane 2), NTERA-2 (Lane 3), U-2 OS (Lane 4), A549 (Lane 5), Mouse Brain (Lane 6) and PC-12 (Lane 7). The blots were probed with Anti-Fe65 Rabbit Polyclonal Antibody (Product # PA1-752, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A ~ 77 kDa band corresponding to Fe65 was observed across cell lines and tissue tested. Additionally ~ 41 kDa band was observed in U-87 MG, A549, and Mouse brain. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), 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 iBlot® 2 Dry Blotting System (Product # IB21001). 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: Immunofluorescence analysis of Fe65 was performed using 70% confluent log phase SH-SY5Y cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Fe65 Rabbit Polyclonal Antibody (Product # PA1-752) 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 cytoplasmic 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: Flow cytometry analysis of Fe65 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 Fe65 Rabbit Polyclonal Antibody (Product # PA1-752, red histogram) or with rabbit isotype control (pink histogram) at 3-5 µg/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 (Product # 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: NULL

PA1-752

Antibody data