A fast Western blot procedure improved for quantitative analysis by direct fluorescence labeling of primary antibodies

Bergendahl, Veit; Glaser, Bryan T.; Burgess, Richard R.

doi:10.1016/s0022-1759(03)00183-2

Cited by 18 publications

(21 citation statements)

References 6 publications

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“…7). The resulting consensus was represented as a ggcTTGa (N) [12][13][14][15][16][17][18][19][20] cCCCAT, where lowercase letters indicate a less highly conserved site. Higher sequence conservation was observed in the Ϫ10 region.…”

Section: Comparison Of Microarray Data With Rt-pcr Results-formentioning

confidence: 99%

“…Therefore, we think our approach provides a "purer" and more complete set (if not all) of genes in the 32 regulon. We have also utilized a new method of quantitative Western blotting (19) to measure the intracellular protein levels of factors. Measuring both the protein level of 32 and the transcriptional levels of 32 -controlled genes as a function of time provides valuable information for exploring the complex network regulated by 32 and gives us an insight into how the 32 protein level regulates the transcriptional level of 32 -controlled genes in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…Mouse mAbs used in this experiment were anti-␣ (4RA2), anti-␤Ј (NT73), anti-70 (2G10), and anti-32 (3RH3). Fluorescence dye, IC5-OSu (Dojindo), was used to label the primary antibodies according to methods described previously (19). The IC5-labeled mAbs, stored at a final concentration of 1 mg/ml, were diluted 1:2000 for use in this experiment.…”

Section: Methodsmentioning

confidence: 99%

“…Quantitation of 32 after Induction-A newly developed quantitative Western blot method (19) was used to monitor the intracellular level of 32 in vivo before and after induction. The ␣-or ␤Ј-subunits of core RNA polymerase were also examined to serve as internal controls because their intracellular levels remain constant under various conditions (21,22).…”

Section: The Construction Of E Coli Strain Mg1655k1-thementioning

confidence: 99%

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The Global Transcriptional Response of Escherichia coli to Induced σ32 Protein Involves σ32 Regulon Activation Followed by Inactivation and Degradation of σ32 in Vivo

Zhao

Liu

Burgess

2005

Journal of Biological Chemistry

Self Cite

129

144

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Escherichia coli core DNA-dependent RNA polymerase consists of four different subunits and has the composition ␣ 2 ␤␤Ј. Core RNA polymerase (E) together with a factor constitutes a holoenzyme complex (E) (1, 2). The holoenzyme complex is able to initiate transcription at specific DNA sequences termed promoters. Since the discovery of 70 36 years ago, numerous factors have been described in E. coli and other prokaryotic organisms (3-7). The seven known E. coli factors are 70 , 54 , 32 , S , F , E , and fecI .The major factor, 70 , is involved in the transcription of the majority of genes in the cell. The other factors are alternative factors that enable RNA polymerase to transcribe genes required for cellular adaptation to changes in the external environment. Each factor recognizes and directs RNA polymerase to a different set of promoters.Among the six known alternative factors, 32 , which is encoded by rpoH (htpR, hin, and fam), was the first minor factor to be discovered in E. coli. For heat shock and some other general stress responses (such as sublethal concentrations of ethanol, viral infection, etc.), transcription initiation is regulated largely by 32 . The first step of the transcription initiation pathway is the binding of 32 to core RNA polymerase to form an E 32 holoenzyme complex. This binding results in the expression of many heat shock proteins (HSPs) 1 that play important roles in protein folding, repair, and degradation under normal and stress conditions. Because 32 plays an important role in heat shock stress, early work on 32 -dependent genes was focused on the induction of a group of genes upon heat shock stress. Most known 32 -dependent genes were identified either by monitoring synthesis rates of individual proteins before and after heat shock on two-dimensional gels (8) or by hybridizing cDNA (generated mRNA from heat-shocked cells) with membrane filters containing an ordered E. coli genomic library (9). However, in response to temperature upshift, the induction of S was shown in a Western blot experiment, and the induction of S -dependent genes was confirmed by using a S -dependent promoter-lacZ fusion approach (10). Also, Taylor and co-workers (11) found 54 -controlled genes are another group of genes that can be induced by heat shock in addition to 32 and E regulons (12). Therefore, although the heat shock response is mainly mediated by 32 , there are some other global gene regulators that increase and turn on genes during the heat shock response. This makes the heat shock stimulon a complicated group of different regulons.Here we report the results of using transcription profiling experiments to identify the 32 regulon in E. coli. Our basic strategy is to minimally perturb steady-state growth (E. coli MG1655 growing exponentially in minimum medium at 37°C) by moderate induction of 32 . We then monitor global RNA transcript abundance changes as a function of time using Affymetrix GeneChip R E. coli antisense genome arrays. This approach allows us to reduce the possibility of induction ...

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Section: Comparison Of Microarray Data With Rt-pcr Results-formentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: The Construction Of E Coli Strain Mg1655k1-thementioning

confidence: 99%

See 2 more Smart Citations

The Global Transcriptional Response of Escherichia coli to Induced σ32 Protein Involves σ32 Regulon Activation Followed by Inactivation and Degradation of σ32 in Vivo

Zhao

Liu

Burgess

2005

Journal of Biological Chemistry

Self Cite

129

144

View full text Add to dashboard Cite

show abstract

“…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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Horseradish‐peroxidase‐conjugated anti‐erythropoietin antibodies for direct recombinant human erythropoietin detection: Proof of concept

Voss

Orie

El-Saftawy

et al. 2020

Drug Testing and Analysis

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Antidoping testing for recombinant human erythropoietin (EPO) is routinely performed by gel electrophoresis followed by western blot analysis with primary and secondary antibodies. The two antibody steps add more than 24 h to the testing time of a purified sample. The aim of this study was to test the concept of using directly horseradish‐peroxidase (HRP)‐conjugated anti‐EPO primary antibody, without the need for a secondary antibody, to reduce the analysis time and eliminate non‐specific cross‐reactivity with secondary antibodies. An in‐house, periodate coupling (R&D systems, clone AE7A5) and three commercially available anti‐human EPO‐HRP conjugates from Genetex, Novus Biologicals and Santa Cruz were evaluated for specificity and sensitivity, using recombinant human EPO standards, negative human urine samples and urine samples from an EPO excretion study. The in‐house anti‐EPO‐HRP conjugate was performed as well as the current two‐step application of unconjugated primary and secondary antibodies used in routine analysis, with comparable specificity and sensitivity. The analysis time was markedly reduced for purified samples from 25 h with the routine method down to 7 h with the in‐house HRP conjugate. Of the three commercially available conjugates tested, only the Santa Cruz anti‐EPO‐HRP conjugate showed comparable specificity but had lower sensitivity to both the in‐house and the antibody combination currently applied routinely. The other two commercially available conjugates (Genetex and Novus Biologicals) did not show any visible bands with the EPO standards. The results clearly demonstrate the potential utility of a directly HRP‐conjugated anti‐EPO antibody to reduce analysis time for EPO in doping control.

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A fast Western blot procedure improved for quantitative analysis by direct fluorescence labeling of primary antibodies

Cited by 18 publications

References 6 publications

The Global Transcriptional Response of Escherichia coli to Induced σ32 Protein Involves σ32 Regulon Activation Followed by Inactivation and Degradation of σ32 in Vivo

The Global Transcriptional Response of Escherichia coli to Induced σ32 Protein Involves σ32 Regulon Activation Followed by Inactivation and Degradation of σ32 in Vivo

Multistrip Western blotting to increase quantitative data output

Horseradish‐peroxidase‐conjugated anti‐erythropoietin antibodies for direct recombinant human erythropoietin detection: Proof of concept

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