The Use of Infrared Fluorescent Dyes in Quantitative Immunoblotting

Abstract

AbstractCentrosomes are microtubule organizing centers that regulate assembly of the mitotic spindle apparatus (1). Because excess centrosomes generate aberrant mitotic spindles, the duplication of centrosomes must be tightly controlled (2). Centrosome duplication requires the activity of the Mps1 protein kinase (3), and Mps1 levels are regulated by proteasome-mediated degradation (4). Mps1 is a Cdk2 substrate, and phosphorylation of Mps1 by Cyclin-dependent kinase 2 (Cdk2) prevents the degradation of Mps1 as part of the mechanism restricting centrosome duplication within the cell cycle (4, 5). Accordingly, inhibition of Cdk2 activity with the small molecule roscovitine, or depletion of Cdk2 activity with cyclin A- or Cdk2-specific siRNAs, all lead to the loss of Mps1 from cen-trosomes and prevent centrosome duplication. However, these different means of inhibiting Cdk2 activity have different consequences for the whole-cell levels of Mps1; roscovitine can result in the near complete loss of Mps1 from the cell (4, 5), while Cdk2-specific siRNAs reduce whole-cell Mps1 levels only by roughly 25% and cyclin A-specific siRNAs have virtually no effect on whole-cell Mps1 levels (4). In addition, inhibition of the proteasome causes a 2.5-fold increase in centrosomal Mps1 levels, but causes only a modest increase in whole-cell Mps1 levels (4).Due to unavoidable differences in sample loading such quantitative comparisons between samples require normalization to a loading control such as β-actin (6), αTubulin (3, 4, 7), or βTubulin (8). When comparing Mps1 between samples our choice has been αTubulin because its levels are not affected by Cdk2 or proteasome activity (3–5). However, Mps1 and αTubulin are present at dramatically different levels and using enzymatic detection methods require dramatically different film exposures (seconds for αTubulin versus minutes for Mps1) that are incompatible with detection on the same blot. This requires that the two proteins be detected on separate blots, introducing an unavoidable source of error and limiting the utility of enzymatic detection methods. In contrast, proteins of vastly different abundance can be detected simultaneously in the same lane of an immunoblot when secondary antibodies conjugated to infrared dyes are used in conjunction with the Odyssey laser scanner (LI-COR, Lincoln, NE). Unlike film detection there is no signal spread in laser scanning detection, and blots can be scanned multiple times without any loss of signal. Therefore, antigens that differ in concentration by orders of magnitude can be simultaneously measured using the same secondary antibody. Furthermore, the Odyssey can detect two differently labeled secondary antibodies at the same time, allowing four or more antigens to be measured simultaneously in the same lane of an immunoblot, provided they are well separated by size and good quality primary antibodies are available. Accordingly, based on its sensitivity and accuracy over large concentration ranges, the LI-COR Odyssey is increasingly used for detection of low abundance proteins and quantitative immunoblotting (3, 4, 6–8).KeywordsCdk2 ActivityCentrosome DuplicationQuantitative ImmunoblottingInhibit Cdk2 ActivityCdk2 SubstrateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.