Anti-Tubulin antibody [YL1/2] - Loading Control (ab6160)

  Western blot - Tubulin antibody [YL1/2] - Loading Control (ab6160)

All lanes : Anti-Tubulin antibody [YL1/2] - Loading Control (ab6160) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 3 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Peroxidase Conjugated AffiniPure Rabbit Anti-Rat IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 50 kDa
Observed band size : 52 kDa (why is the actual band size different from the predicted?)
Additional bands at : 85 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 8 minutes

  Western blot - Tubulin antibody [YL1/2] - Loading Control (ab6160)

Lanes 1 & 3 : Anti-Tubulin antibody [YL1/2] - Loading Control (ab6160) at 1/5000 dilution
Lanes 2 & 4 : Anti-Tubulin antibody [YL1/2] - Loading Control (ab6160) at 1/10000 dilution

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 3 : BALB/3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab7901)
Lane 4 : BALB/3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab7901)

Lysates/proteins at 20 µg per lane.


Predicted band size : 50 kDa
Observed band size : 52 kDa (why is the actual band size different from the predicted?)
Additional bands at : 17 kDa,34 kDa,80 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 10 seconds

  Immunocytochemistry/ Immunofluorescence - Tubulin antibody [YL1/2] - Loading Control (ab6160)

This image was kindly supplied as part of the review submitted by Marko Kallio. Ab6160 was used for immunofluorescence on male rat testis samples in order to visualize microtubules of meiotically deviding cells. The samples were fixed with 2% paraformaldehyde and 0.8% glutaraldehyde and the antibody was used at a dilution 1:2500 (red - tubulin, blue - DNA stained with DAPI).

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Tubulin antibody [YL1/2] - Loading Control (ab6160)

ab6160 staining mouse prostate tissue sections by IHC-P.  The tissue was serial sectioned at 6 microns, formaldehyde fixed and subjected to heat mediated antigen retrieval prior to blocking in 3% peroxidase for 5 minutes at 27°C.  The primary antibody was diluted 1/500 and incubated with the sample for 16 hours at 4°C.  A Cy5® conjugated goat anti-rat antibody was used as the secondary.

This image is courtesy of an anonymous Abreview

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  Immunocytochemistry/ Immunofluorescence - Tubulin antibody [YL1/2] - Loading Control (ab6160)

ab6160 at 1/1000 dilution staining Tubulin in human WBC cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed in acetone and then blocked in 5% serum for 1 hour at 25°C. No permeabilization was done. The primary antibody was used at 1/1000 dilution in PBS-Tween and incubated with sample at 4°C for 16 hours. An Alexa Fluor® 594 conjugated goat polyclonal to rat IgG was used as secondary at 1/500 dilution.

This image is courtesy of an anonymous Abreview.

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  Immunohistochemistry (Frozen sections) - Tubulin antibody [YL1/2] - Loading Control (ab6160)

ab6160 at a 1/200 staining Tubulin in mouse liver tissue sections by Immunohistochemistry (frozen sections) incubated for 9 hours at +4°C. Fixed in formaldehyde, permeabilized using 0.2% Triton X-100. Blocked using 2% BSA for 30 minutes at 20°C. Secondary used at a 1/200 dilution polyclonal Goat anti-rat IgG conjugated to Alexa Fluor 555.

This image is courtesy of an anonymous abreview.

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  Immunocytochemistry/ Immunofluorescence - Tubulin antibody [YL1/2] - Loading Control (ab6160)

ab6160 staining Tubulin in mouse MEF cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed with 2% PFA  and 96% Ethanol. Samples were incubated with primary antibody (1/2000 in 0.1% Saponin/1% BSA/PBS) for 1 hour. A Cyt3^®-conjugated goat polyclonal to rat IgG (H&L) was used at dilution at 1/500 as secondary antibody. Red staining in the image represents Tubulin, whereas the green one resembles gamma-tubulin.

This image is a courtesy of Anonymous Abreview

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  Flow Cytometry-Anti-Tubulin antibody [YL1/2] - Loading Control(ab6160)

Overlay histogram showing HeLa cells stained with ab6160 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab6160, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rat IgG (H+L) (ab98386) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rat IgG2a [aRTK2758] (ab18450, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.