- PA5-19489 - Invitrogen Antibodies TUBA1C antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of acetylated alpha-Tubulin was performed using whole cell lysates from cells left untreated (DMSO only) or cells treated with 0.3uM or 3uM Trichostatin A (TSA) for 16 hours. Antigen-antibody complexes were formed by incubating 500 µg of lysate with 3 µg of an Acetyl Lysine monoclonal antibody (Product # MA1-2021) overnight on a rocking platform at 4°C. The immune complexes were captured on 50 µL Protein A/G Agarose (Product # 20421), washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer (Product # 39000). Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBS-0.1%Tween for at least 1 hour. The membrane was probed with an alpha-Tubulin polyclonal antibody (Product # PA5-19489) at a dilution of 1:1000 overnight rotating at 4°C, washed in TBST, and probed with Clean-blot IP Detection Reagent (Product # 21230) at a dilution of 1:2000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34087).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of A-431 (Lane 1), COS-7 (Lane 2), MDCK (Lane 3), NIH/3T3 (Lane 4), HT-29 (Lane 5), Neuro-2a (Lane 6) and tissue extract of Rat Brain (Lane 7). The blot was probed with Rabbit Anti-alpha Tubulin Polyclonal Antibody (Product # PA5-19489, 0.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 52 kDa band corresponding to alpha Tubulin was observed across the cell lines and tissue tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), 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)
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Experimental details: Immunofluorescence analysis of alpha Tubulin was performed using 70% confluent log phase LNCaP 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 Alpha Tubulin Rabbit Polyclonal Antibody (Product # PA5-19489) 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) at 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 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: Immunofluorescence analysis of alpha Tubulin was performed using 70% confluent log phase NIH/3T3 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 Alpha Tubulin Rabbit Polyclonal Antibody (Product # PA5-19489) 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) at 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 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)
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Experimental details: Immunofluorescent staining of HeLa cells using Product # PA5-19489, anti-Alpha Tubulin antibody. The cells were fixed with methanol (100%) for 5 minutes and exposed to the primary antibody at a concentration of 1 µg/mL for 1 hour at room temp. The secondary antibody was a 448 fluorescence conjugated Goat anti-rabbit IgG (green) at a dilution of 1:1000. A WGA- 594 fluorescent conjugated stain was used to label plasma membranes (red) and the nuclei stain was DAPI (blue).

Supportive validation

Supportive validation

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. Partial rescue of mitotic defects in RT112 cells upon mild overexpression of TACC3. ( a ) Western blot analysis of TACC3 expression level from different promoters (pCMV versus pTACC3 or pPGK promoter) using alpha-GFP or alpha-TACC3 antibodies. alpha-Tubulin was used as the loading control. pCMV, promoter of cytomegalovirus; pTACC3, promoter of TACC3; pPGK, promoter of phosphoglycerate kinase. ( b ) Representative micrographs of the amount of GFP-TACC3 overexpression under pCMV and pTACC3. Scale bar, 10 um. ( c , d ) RT112 cells co-expressing H2B-mCherry with GFP or GFP-TACC3 under pTACC3, synchronized by a double thymidine block. Movies were started 3 h post-release and imaged every 3 min for the next 12 h. Proportions of cells with various mitotic phenotypes are shown ( c ). Categories are as described for all other figures. Cumulative histograms of mitotic progression of all cells ( d ). Prometaphase-metaphase timing is shown for the conditions indicated. GFP: n = 3, 111 cells. GFP-TACC3: n = 3, 95 cells. The full mitotic progression dataset is shown in the electronic supplementary material, figure S1.

PA5-19489