A tris (2-carboxyethyl) phosphine (TCEP) related cleavage on cysteine-containing proteins

Liu, Peiran; O’Mara, Brian W.; Warrack, Bethanne M.; Wu, Wei; Huang, Yunping; Zhang, Yihong; Rulin, Zhao; Lin, Mei Huey; Ackerman, Michael S.; Hocknell, Peter; Chen, Guodong; Tao, Liyuan; Rieble, Siegfried; Wang, Jack; Wang‐Iverson, David; Tymiak, Adrienne A.; Grace, Michael J.; Russell, Reb J.

doi:10.1016/j.jasms.2010.01.016

Cited by 65 publications

(45 citation statements)

References 43 publications

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“…The molecular mechanism is unknown, although the Laemmli buffer is known to selectively break disulfide bonds and denature the proteins while solubilizing them. Dithiothreitol 27 and TCEP 28 have an additional proteolytic effect, which has not been described for 2-ME. There may be, however, other unknown mechanisms by which 2-ME facilitates the detection of antigens in the tissue.…”

Section: Discussionmentioning

confidence: 81%

A 2-Step Laemmli and Antigen Retrieval Method Improves Immunodetection

Scalia

Gendusa²,

Cattoretti³

2016

Applied Immunohistochemistry &Amp; Molecular Morphology

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Detection by immunohistochemistry of antigens relies on reproducibly optimal preanalytical and analytical variables such as fixation conditions, antigen retrieval (AR), and the resolutive power of the detection system. There is a need to improve immunodetection on routinely fixed and embedded material, particularly for scarcely represented but relevant antigens. We devised a 2-step method and applied it to a panel of antigens of common use for diagnosis, prognosis, individualized therapy use, or research. The first step consists of a 10 minutes. Incubation at 95°C with a modified Laemmli extraction buffer. This was followed by a traditional AR method. Detection of the vast majority of antigens was improved over a simple AR with preservation of tissue integrity, as shown by quantitative image analysis. The mechanism underlying the improved detection may be controlled denaturation followed by heat-mediated retrieval, a method we dubbed "antigen relaxing" and which will improve routine detection of scarce antigens in formalin-fixed, paraffin-embedded material.

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Section: Discussionmentioning

confidence: 81%

A 2-Step Laemmli and Antigen Retrieval Method Improves Immunodetection

Scalia

Gendusa²,

Cattoretti³

2016

Applied Immunohistochemistry &Amp; Molecular Morphology

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“…The aggregation was probably because the exposed –SH group formed non-specific inter-molecular disulfide bonds. Consistent with this prediction, addition of strong reducing agents such as 5 mM DTT (dithiothreitol) or 2 mM TCEP (Tris 2-carboxyethyl phosphine) to the purification buffers (Liu et al, 2010) had shifted most of the aggregated protein to the monomeric position.…”

Section: Resultsmentioning

confidence: 63%

Designing a nine cysteine-less DNA packaging motor from bacteriophage T4 reveals new insights into ATPase structure and function

et al. 2014

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The packaging motor of bacteriophage T4 translocates DNA into the capsid at a rate of up to 2000 bp/s. Such a high rate would require coordination of motor movements at millisecond timescale. Designing a cysteine-less gp17 is essential to generate fluorescently labeled motors and measure distance changes between motor domains by FRET analyses. Here, by using sequence alignments, structural modeling, combinatorial mutagenesis, and recombinational rescue, we replaced all nine cysteines of gp17 and introduced single cysteines at defined positions. These mutant motors retained in vitro DNA packaging activity. Single mutant motors translocated DNA molecules in real time as imaged by total internal reflection fluorescence microscopy. We discovered, unexpectedly, that a hydrophobic or nonpolar amino acid next to Walker B motif is essential for motor function, probably for efficient generation of OH− nucleophile. The ATPase Walker B motif, thus, may be redefined as “β-strand (4–6 hydrophobic-rich amino acids)–DE-hydrophobic/nonpolar amino acid”.

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“…However, an advantage of DIGE is that this method is able to detect differential expression of post-translationally modified proteins as well as different isoforms of proteins by resolving spots at different pI and molecular weight. These isoforms may not be distinguished with isobaric approaches, since labeling is completed at the peptide level and most peptide sequences are identical among a group of isoforms [29]. Therefore, the use of different proteomic methods is advisable to obtain complementary datasets [30] to functionally interpret the effect of each diet in the whole liver proteome.…”

Section: Discussionmentioning

confidence: 99%

Liver Proteome Changes Induced by a Short-Term High-Fat Sucrose Diet in Wistar Rats

Bondia‐Pons¹,

Boqué²,

Paternain³

et al. 2011

Lifestyle Genomics

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Background/Aims: The aim of this study was to gain insight into those proteins that might be involved in the early stages of liver fat accumulation as a consequence of a different fat versus simple sugar dietary intake. Methods: Forty-five male Wistar rats were randomly distributed into four dietary groups: a starch-rich control diet (CD; n = 10), a high-fat diet (n = 12), a high-sucrose diet (n = 11), and a high-fat sucrose diet (HFSD; n = 12) for 5 weeks. A comparative analysis by 2D-DIGE and LC-ESI-MS/MS was performed to characterize the liver protein expression profiles due to the three obesogenic diets. Results: Ten out of 17 proteins whose expression levels were altered by >1.25-fold were identified. Four proteins (Hspa8, Hspa9, Ca3, and Cat) were differentially expressed after the HFSD period compared to CD. The heat shock proteins (Hspa8 and Hspa9) resulted significantly downregulated in liver from rats fed HFSD versus CD (p < 0.05). The results were confirmed by Western blot. Conclusions: This descriptive study might be useful for further studies aiming at understanding the mechanisms by which diets rich in both fat and sugar affect the initiation of hepatic steatosis.

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A tris (2-carboxyethyl) phosphine (TCEP) related cleavage on cysteine-containing proteins

Cited by 65 publications

References 43 publications

A 2-Step Laemmli and Antigen Retrieval Method Improves Immunodetection

A 2-Step Laemmli and Antigen Retrieval Method Improves Immunodetection

Designing a nine cysteine-less DNA packaging motor from bacteriophage T4 reveals new insights into ATPase structure and function

Liver Proteome Changes Induced by a Short-Term High-Fat Sucrose Diet in Wistar Rats

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