HRP Anti-alpha Tubulin antibody [DM1A] - Loading Control

• WB

Project

Western blot - HRP Anti-alpha Tubulin antibody [DM1A] - Loading Control (AB40742)

All lanes:

Western blot - HRP Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab40742) at 1/5000 dilution

Lane 1:

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

Lane 2:

Western blot - NIH/3T3 whole cell lysate (<a href='/en-us/products/cell-lysates/nih-3t3-whole-cell-lysate-ab7179'>ab7179</a>) at 10 µg

Lane 3:

Brain (Rat) Tissue Lysate at 10 µg

Lane 4:

Xenopus Laevis Protein Extracts (Stg 8) at 10 µg

Predicted band size: 50 kDa

Observed band size: 57 kDa

false

Exposure time: 2min

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - HRP Anti-alpha Tubulin antibody [DM1A] - Loading Control (AB40742)

IHC image of alpha Tubulin staining in a section of formalin-fixed paraffin-embedded normal human colon tissue*, performed on a Leica BOND^TM. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab40742, 1/100 dilution, for 15 mins at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

• WB

Lab

Western blot - HRP Anti-alpha Tubulin antibody [DM1A] - Loading Control (AB40742)

Phosphorylation level of S757 is reduced during starvation (PMID : 28835610, 21258367). This blot was developed using a higher sensitivity ECL substrate.

Blocking/Diluting buffer and concentration : 5% NFDM/TBST.

Exposure time : 59 seconds.

All lanes:

Western blot - Anti-ULK1 (phospho S757) antibody [EPR22265-9] (<a href='/en-us/products/primary-antibodies/ulk1-phospho-s757-antibody-epr22265-9-ab229909'>ab229909</a>) at 1/1000 dilution

Lane 1:

Untreated MEF (mouse embryonic fibroblast (immortalized)), whole cell lysate at 20 µg

Lane 2:

MEF cells were cultured in amino acid and serum-free, glucose-containing starvation buffer (EBSS) for 16 hours, whole cell 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-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 150 kDa,50 kDa

false

Exposure time: 59s