Anti-alpha Tubulin antibody - Loading Control

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody - Loading Control (AB4074)

ab4074 staining alpha Tubulin in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab4064 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 100% methanol (5 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody - Loading Control (AB4074)

Immunocytochemistry of SisNPFR-C6E8 cell line. Cells were prefixed in freshly prepared 4% paraformaldehyde (PFA) in F12 K culture medium with 10% fetal bovine serum and Geneticin. Fixed cells were permeabilized in PBST (0.25% Triton X-100 in PBS) for 5 min and blocked at 4°C overnight in 5% normal goat serum. The cells were then incubated at 4°C overnight with ab4074 (1 : 50). Cover slips were placed onto slides and mounted with VectaShield containing DAPI.

Image from PLoS One. 2014; 9(10): e109590. Fig 5o,doi: 10.1371/journal.pone.0109590 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• WB

Ap

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB4074)

All lanes:

Western blot - Anti-alpha Tubulin antibody - Loading Control (ab4074) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (ab27252) at 20 µg

Lane 2:

HEK-293 (Human) Whole Cell Lysate (ab52256) at 20 µg

Lane 3:

Western blot - NIH/3T3 whole cell lysate (<a href='/en-us/products/cell-lysates/nih-3t3-whole-cell-lysate-ab7179'>ab7179</a>) at 20 µg

Lane 4:

PC-12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957) at 20 µg

Lane 5:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (ab27252) at 20 µg with Human alpha Tubulin peptide (<a href='/en-us/products/unavailable/human-alpha-tubulin-peptide-ab23537'>ab23537</a>)

Lane 6:

HEK-293 (Human) Whole Cell Lysate (ab52256) at 20 µg with Human alpha Tubulin peptide (<a href='/en-us/products/unavailable/human-alpha-tubulin-peptide-ab23537'>ab23537</a>)

Lane 7:

Western blot - NIH/3T3 whole cell lysate (<a href='/en-us/products/cell-lysates/nih-3t3-whole-cell-lysate-ab7179'>ab7179</a>) at 20 µg with Human alpha Tubulin peptide (<a href='/en-us/products/unavailable/human-alpha-tubulin-peptide-ab23537'>ab23537</a>)

Lane 8:

PC-12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957) at 20 µg with Human alpha Tubulin peptide (<a href='/en-us/products/unavailable/human-alpha-tubulin-peptide-ab23537'>ab23537</a>)

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution

Predicted band size: 50 kDa

false

• WB

Ap

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB4074)

All lanes:

Western blot - Anti-alpha Tubulin antibody - Loading Control (ab4074) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (ab27252) at 10 µg

Lane 2:

NIH/3T3 (Mouse) Whole Cell Lysate (ab52956) at 10 µg

Lane 3:

PC-12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957) at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution

Predicted band size: 50 kDa

false

• WB

Project

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB4074)

ab4074 cross-reacts strongly with Mouse 3T3 lysate and shows weak cross-reactivity with Rat liver, Cow, Chicken and CHO lysates.

All lanes:

Western blot - Anti-alpha Tubulin antibody - Loading Control (ab4074) at 1 µg/mL

Lane 1:

Mouse 3T3 lysate at 20 µg

Lane 2:

Rat liver lysate at 20 µg

Lane 3:

Cow cell lysate at 20 µg

Lane 4:

Chicken cell lysate at 20 µg

Lane 5:

CHO lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab6721'>ab6721</a>) at 1/5000 dilution

Predicted band size: 50 kDa

Observed band size: 50 kDa

true

Exposure time: 30s