- 711212 - Invitrogen Antibodies VIM antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HeLa (Lane 1), HeLa treated with Paclitaxel (100nM for 20 Hours) (Lane 2), COS-7 (Lane 3), COS-7 treated with Paclitaxel (100nM for 20 Hours) (Lane 4), L6 (Lane 5) and L6 treated with Paclitaxel (100nM for 20 Hours) (Lane 6). The blots were probed with Anti-Vimentin (pS56) Recombinant Rabbit Polyclonal Antibody (Product # 711212, 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 54 kDa band corresponding to Vimentin (pS56) was observed. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), 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® 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 analysis was performed on Whole cell extracts (30 µg lysate) of HeLa (Lane 1), HeLa treated with Paclitaxel (100nM for 20 Hours) (Lane 2), COS-7 (Lane 3), COS-7 treated with Paclitaxel (100nM for 20 Hours) (Lane 4), L6 (Lane 5) and L6 treated with Paclitaxel (100nM for 20 Hours) (Lane 6). The blots were probed with Anti-Vimentin (pS56) Recombinant Rabbit Polyclonal Antibody (Product # 711212, 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 54 kDa band corresponding to Vimentin (pS56) was observed. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), 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® 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: For immunofluorescence analysis, SW480 cells were fixed and permeabilized for detection of endogenous Vimentin pS56 using Anti-Vimentin pS56 Recombinant Rabbit Polyclonal Antibody (Product # 711212, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Detection and localization of Vimentin pS56 (green) in the cytoplasm can be clearly observed in cells treated with Paclitaxel (100 nM, 20 h). Antibody specificity was demonstrated by competition with the Vimentin pS56 peptide, which results in loss of signal. No competition was observed with the non-phospho peptide. The images were captured at 60X magnification. The antibody detected a nuclear signal for Vimentin pS56 in untreated cells, which was also competed out specifically by the phospho peptide (data not shown). Although vimentin is predominantly cytoplasmic, its nuclear localization has been reported. The role of S56 phosphorylation in the nucleus however has not been established to date.

711212