Multistrip Western blotting to increase quantitative data output

Abstract

The qualitative and quantitative measurement of protein abundance and protein modification states are essential in understanding their role in diverse cellular processes. Traditional Western blotting technique, though sensitive, is prone to produce substantial errors and is not readily adapted to highthroughput technologies. We propose a modified immunoblotting procedure, which is based on simultaneous transfer of proteins from multiple gel-strips onto the same membrane, and is compatible with any conventional gel electrophoresis system. As a result, the data output per single blotting cycle can readily be increased up to ten-fold. In contrast to the traditional "one protein detection per electrophoresis cycle", this procedure allows simultaneous monitoring of up to nine different proteins. In addition to maintaining the ability to detect picogram quantities of protein, the modified system substantially improves data accuracy by reducing signal errors by two-fold. Multi-strip Western blotting procedure allows making statistically reliable side-by-side comparisons of different or repeated sets of data. Compared to the traditional methods, this approach provides a more economical, reproducible and effective procedure, facilitating the generation of large amounts of high-quality quantifiable data.

Human Milk Hyaluronan Enhances Innate Defense of the Intestinal Epithelium

“…Separated proteins were transferred at 4°C to PVDF membrane (Millipore Corp., Billerica, MA) by an electroblotting apparatus (Bio-Rad) at 110 V for 60 min. In cases where analysis of numerous replicate samples or multiple protein targets of differing molecular weight was required, a multistrip Western blotting protocol was used to increase quantitative output and improve signal consistency (54). PVDF membranes were blocked in Odyssey Blocking Buffer (LI-COR) diluted to 50% concentration in Trisbuffered saline (TBS) for 60 m. The membrane was incubated with rabbit polyclonal antibody against H␤D2 (Abcam, Cambridge, MA) at 1:750 in the blocking buffer, and the primary antibody was followed by biotin-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at 1:30,000 in blocking buffer and finally horseradish peroxidase (HRP)-conjugated streptavidin (GE Healthcare) at 1:100,000 in TBS alone.…”

Specific-sized Hyaluronan Fragments Promote Expression of Human β-Defensin 2 in Intestinal Epithelium

“…Separated proteins were transferred at 4°C to PVDF membrane with an electroblotting apparatus (Bio-Rad) at 100 V for 45 min. In cases where analysis of numerous replicate samples or multiple protein targets of differing molecular weight were required, the multistrip Western blotting protocol developed by Aksamitiene et al (37) was used to increase quantitative output and improve signal consistency. PVDF membranes were air-dried at room temperature for 60 min prior to blocking with Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE) diluted to 50% concentration in Tris-buffered saline.…”

The Experimental Side of Parameter Estimation

“…However, it is possible to run several gels (at the same time), then cut the band of interest and transfer them in one go onto the same membrane. This technique is called multi-strip western blotting [3].…”