High-Efficiency Blotting of Proteins of Diverse Sizes Following Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

Bolt, Mark W.; Mahoney, Paul A.

doi:10.1006/abio.1997.2061

Cited by 136 publications

(71 citation statements)

References 16 publications

(2 reference statements)

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“…Cell extracts were normalized to cell number and run on either 8% or 10% SDS-PAGE gels and transferred to PVDF membrane (Immobilion, Millipore) by wet transfer using Bolt and Mahoney buffer (40 mM Tris, 10 mM sodium acetate, 2 mM EDTA, 0.05% SDS, 20% MeOH) at 1A for 45 min [81]. Blots were blocked with 1% non-fat milk in TBS (150 mM NaCl, 10 mM Tris, pH 8.0, 0.05% Tween 20) for 1 h at room temperature.…”

Section: Antibody Purification and Immunoblottingmentioning

confidence: 99%

Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle

McNairn

Okuno

Misteli

et al. 2005

Experimental Cell Research

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In yeast, the Origin Recognition Complex (ORC) is bound to replication origins throughout the cellcycle, but in animal cells, there are conflicting data as to whether and when ORC is removed from chromatin. We find ORC1, 2 and ORC4 to be metabolically stable proteins that co-fractionate with chromatin throughout the cell-cycle in Chinese hamster fibroblasts. Since cellular extraction methods cannot directly examine the chromatin binding properties of proteins in vivo, we examined ORC:chromatin interactions in living cells. Fluorescence loss in photobleaching (FLIP) studies revealed ORC1 and ORC4 to be highly dynamic proteins during the cell-cycle with exchange kinetics similar to other regulatory chromatin proteins. In vivo interaction with chromatin was not significantly altered throughout the cell-cycle, including S-phase. These data support a model in which ORC subunits dynamically interact with chromatin throughout the cell-cycle.

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Section: Antibody Purification and Immunoblottingmentioning

confidence: 99%

Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle

McNairn

Okuno

Misteli

et al. 2005

Experimental Cell Research

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“…Proteins in cell lysates were separated by a 10% SDS-polyacrylamide gel electrophoresis according to Laemmli (Laemmli, 1970) and electro-transferred onto a polyvinylidene difluoride membrane according to Bolt and Mahoney (Bolt and Mahoney, 1997). The membrane was blocked with 1% (w/v) skim milk, 0.1% tween (v/v) in Tris-buffered saline, pH 7.4 for 30 min and incubated with specific primary antibodies at 4 o C overnight and secondary antibody conjugated-HRP at room temperature for 2h.…”

Section: Western Blottingmentioning

confidence: 99%

In vitro and In vivo Antitumor Activity of Tiliacorinine in Human Cholangiocarcinoma

Janeklang

Nakaew

Vaeteewoottacharn³

et al. 2014

Asian Pacific Journal of Cancer Prevention

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“…electrophoresis and immunoblotting procedures that involve many factors: sampling variance [30], quality and age of the gel (Fig. 2S in the Supplement), composition of buffer systems [19,20,[31][32][33], type of blot module, materials for electroblotting (e.g. firmness of filter paper, shape of fiber pads, type of the membrane support (nitrocellulose or PVDF) [34,35]), optimized time of protein transfer onto the membrane [19,22], quality and dilution of antibodies to optimize signal-to-noise ratio, membrane washing [21], signal detection [26] and normalization of raw data [36].…”

Section: Quality Versus Quantity Problemmentioning

confidence: 99%

“…The processing of such high-throughput data is a costly, timeconsuming multi-step procedure prone to systematic or random errors. Therefore improvements to existing experimental methods are desirable, which provide cheaper, faster and better detection of proteins [19][20][21][22][23][24].…”

Section: Introductionmentioning

confidence: 99%

Multistrip Western blotting to increase quantitative data output

et al. 2007

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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.

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High-Efficiency Blotting of Proteins of Diverse Sizes Following Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis

Cited by 136 publications

References 16 publications

Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle

Chinese hamster ORC subunits dynamically associate with chromatin throughout the cell-cycle

In vitro and In vivo Antitumor Activity of Tiliacorinine in Human Cholangiocarcinoma

Multistrip Western blotting to increase quantitative data output

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