- PA5-29131 - Invitrogen Antibodies SMC3 antibody

Cubeñas-Potts C, Srikumar T, Lee C, Osula O, Subramonian D, Zhang XD, Cotter RJ, Raught B, Matunis MJ
Proteomics 2015 Feb;15(4):763-72

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using SMC3 Polyclonal Antibody (Product # PA5-29131). Sample (30 µg of whole cell lysate). Lane A: NIH-3T3. Lane B: JC. Lane C: BCL-1. 5% SDS PAGE. SMC3 Polyclonal Antibody (Product # PA5-29131) diluted at 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using SMC3 Polyclonal Antibody (Product # PA5-29131). Sample (30 µg of whole cell lysate). Lane A: HepG2.5% SDS PAGE. SMC3 Polyclonal Antibody (Product # PA5-29131) diluted at 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using SMC3 Polyclonal Antibody (Product # PA5-29131). Sample (30 µg of whole cell lysate). Lane A: PC-12. 5% SDS PAGE. SMC3 Polyclonal Antibody (Product # PA5-29131) diluted at 1:2,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of SMC3 was achieved by transfecting HeLa with SMC3 specific siRNAs (Silencer® select Products # s17427 s17426). Western blot analysis (Fig. a) was performed using whole cell extracts from the SMC3 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with SMC3 Polyclonal Antibody (Product # PA5-29131, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25ug/ml, 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 SMC3. (Note: Few uncharacterized bands were observed at ~ 60, ~35 kDa).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-SMC3 Polyclonal Antibody (Product # PA5-29052) and a 160kDa band corresponding to SMC3 was observed in all the tested cell and tissue models. Modified whole cell lysate (1% SDS) (30ug lysate) of A-431 (Lane 1), HeLa (Lane 2), MCF7 (Lane 3), A549 (Lane 4), U-87 MG (Lane 5), SH-SY5Y (Lane 6), HCT 116 (Lane 7), Jurkat (Lane 8) and tissue extract (30ug lysate) of Mouse Liver (Lane 9) were electrophoresed using NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # LC2001) by wet transfer. The blot was probed with the primary antibody (0.5µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP conjugate (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of SMC3 in paraformaldehyde-fixed HeLa cells using a SMC3 polyclonal antibody (Product # PA5-29131) at a 1:200 dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of SMC3 was performed using 70% confluent log phase MCF7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with SMC3 Polyclonal Antibody (Product # PA5-29131) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of paraffin-embedded Cal27 Xenograft, using SMC3 (Product # PA5-29131) antibody at 1:100 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous SMC3 protein using Anti-SMC3 Antibody: ChIP was performed using Anti-SMC3 Rabbit Polyclonal Antibody (Product # PA5-29131, 5 µg) on sheared chromatin from HeLa cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to selected chromosomal positions: Chr 9 (11244 - 11320), Chr 17 (7944729-794507) and Chr 12 (53746435-53746713) and SAT2 satellite repeats. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

Supportive validation

Supportive validation

PA5-29131