Quantitative dot blot analysis (QDB), a versatile high throughput immunoblot method

Abstract

Lacking access to an affordable method of high throughput immunoblot analysis for daily use remains a big challenge for scientists worldwide. We proposed here Quantitative Dot Blot analysis (QDB) to meet this demand. With the defined linear range, QDB analysis fundamentally transforms traditional immunoblot method into a true quantitative assay. Its convenience in analyzing large number of samples also enables bench scientists to examine protein expression levels from multiple parameters. In addition, the small amount of sample lysates needed for analysis means significant saving in research sources and efforts. This method was evaluated at both cellular and tissue levels with unexpected observations otherwise would be hard to achieve using conventional immunoblot methods like Western blot analysis. Using QDB technique, we were able to observed an age-dependent significant alteration of CAPG protein expression level in TRAMP mice. We believe that the adoption of QDB analysis would have immediate impact on biological and biomedical research to provide much needed high-throughput information at protein level in this “Big Data” era.

3D association of ER, PR and Her2 at protein levels to aid sub-grouping of breast cancer patients in routine clinical practice

“…The QDB process was described elsewhere with minor modifications 16,17 . In brief, the final concentration of the FFPE tissue lysates was adjusted to 0.25 μg/μl for Her2 and Ki67 and 0.125 μg/μl for ER and PR, and 2 μl/unit was used for QDB analysis in triplicate.…”

“…Recently, a high throughput immunoblot method, Quantitative Dot Blot analysis (QDB), was developed in the company 16,17 . This method provides a feasible alternative for absolute quantitation of tissue biomarkers in FFPE samples to SRM-MS method, which so far is limited to only a few proteins 11–13,15 .…”

Combined use of absolutely quantitated cyclin D1 and Ki67 protein levels to improve prognosis of Luminal-like breast cancer

“…The QDB process was described previously with minor modifications [8][9][10][11]. In brief, FFPE tissue lysates (about 0.35 g/unit) and MCF-7 cell lysate (about 0.08 g/unit) were applied onto the QDB plate at 2 l/unit in triplicate.…”

Developing a routine lab test for absolute quantification of Her2 in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tissues using Quantitative Dot Blot (QDB) method

“…Recently, Quantitative Dot Blot (QDB) method was developed as a novel high throughput immunoassay platform for absolute quantitation of a specific protein at tissue level [11][12][13][14]. While distinctive in principle from Enzymatic linked immunosorbent assay (ELISA), QDB method nonetheless shares the same convenience, objectivity and consistency with ELISA in action.…”

“…Samples scored as 2 + are defined as equivocal. For these samples, FISH analysis is used to further differentiate Her2 + from Her2- [10,11].…”