The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/5000. Detects a band of approximately 57 kDa (predicted molecular weight: 50 kDa).
1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Microtubules are involved in a wide variety of cellular activities ranging from mitosis and transport events to cell movement and the maintenance of cell shape. Tubulin itself is a globular protein which consists of two polypeptides (alpha and beta tubulin). Alpha and beta tubulin dimers are assembled to 13 protofilaments that form a microtubule of 22 nm diameter. Tyrosine ligase adds a C-terminal tyrosin to monomeric alpha tubulin. Assembled microtubules can again be detyrosinated by a cytoskeleton associated carboxypeptidase. Detyrosinated alpha tubulin is referred to as Glu-tubulin. Another post-translational modification of detyrosinated alpha tubulin is C-terminal polyglutamylation which is characteristic for microtubules in neuronal cells and the mitotic spindle.
Alpha tubulin is not suitable as a loading control in adipose tissue as expression of tubulin in adipose tissue is very low ( Spiegelman and Farmer, Cell, 1982, 29(1): 53-60, "in cells undergoing adipose differentiation actin synthesis decreases by 90%").
IHC image of alpha Tubulin staining in a section of formalin-fixed paraffin-embedded normal human colon tissue*, performed on a Leica BONDTM. 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