The Use of Monoclonal Antibodies and Limited Proteolysis in Elucidation of Structure—Function Relationships in Proteins

Wilson, John E.

doi:10.1002/9780470110560.ch4

Cited by 50 publications

(8 citation statements)

References 197 publications

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“…Here, we probe these putative conformational changes by comparing limited proteolysis of repressed wt-TRCF and derepressed mutants (TRCF-D2*** and TRCF-D7***) of TRCF. Limited proteolysis has long been used to probe the conformational dynamics of proteins in solution, since proteases such as trypsin or chymotrypsin tend to cleave unstructured, dynamic segments of polypeptides rather than well structured regions 17. We find that the repressed wt-TRCF is highly resistant to proteolysis, while the derepressed mutants are dramatically sensitized.…”

Section: Introductionmentioning

confidence: 87%

Derepression of Bacterial Transcription-Repair Coupling Factor Is Associated with a Profound Conformational Change

Srivastava

Darst

2011

Journal of Molecular Biology

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Transcription Repair Coupling Factor (TRCF, the product of the mfd gene) is a widely conserved bacterial protein that couples DNA repair with transcription. TRCF recognizes RNA polymerase stalled at a non-coding lesion in the DNA template strand, uses the energy from ATP hydrolysis to disrupt the transcription complex, and stimulates DNA repair by recruiting UvrA, a component of the nucleotide excision repair machinery, to the site. TRCF is a large (130 kDa), multi-functional protein with a complex structure/function relationship comprising a compact arrangement of eight structured domains linked by flexible linkers. Through a conserved, intramolecular, interdomain interaction, TRCF is held in a conformation in which its enzymatic activities (ATPase activity, DNA translocase activity) are strongly repressed. Disruption of the repressive interdomain interaction by amino acid substitutions within the interface derepresses ATPase and DNA translocase activities. In this work, we have shown that derepressed TRCF mutants are dramatically sensitized to limited proteolysis compared with repressed TRCF, pointing to an altered conformational state. Analysis of the protease cleavage sites mapped onto the structure of the repressed TRCF conformation indicate that: 1) The cleavage sites tend to cluster at linkers connecting the TRCF structured domains, and 2) Many of the cleavage sites sensitized in the derepressed TRCF are partially or completely buried to protease access in the repressed TRCF structure. We conclude that TRCF derepression is associated with profound conformational changes that primarily involve a reorganization of the interdomain interactions.

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Section: Introductionmentioning

confidence: 87%

Derepression of Bacterial Transcription-Repair Coupling Factor Is Associated with a Profound Conformational Change

Srivastava

Darst

2011

Journal of Molecular Biology

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“…EDTA solutions are more effective than citrate buffer for some antigens,63 – 66 but the unmasking effect of calcium buffered solutions cannot be recognised for other markers 67 68…”

Section: Antigen Retrievalmentioning

confidence: 99%

Technical pitfalls potentially affecting diagnoses in immunohistochemistry

2008

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Result of the immunohistochemical reactions routinely used in diagnostic surgical pathology should be properly interpreted, since false results, related to technical and interpretative pitfalls may lead to incorrect diagnosis. The main sources of such pitfalls are reviewed, analytically described and related to different steps (fixation, tissue processing and embedding, decalcification, antigen retrieval) which may affect the accuracy of immunohistochemistry. In addition, the presence of endogenous enzyme activity, improper binding of avidin to endogenous biotin, incorrect use of antibodies, chromogen and detection systems, as well as incorrect interpretation may produce unreliable data. The high frequency and extension of such pitfalls make mandatory the use of internal and external controls and adoption of cross-validation programmes. The present study, supported by an extensive review of the related literature, is intended as a guideline leading to proper interpretation of immunohistochemical data, an essential component of the diagnostic process. Experience on the antigen retrieval procedures for different antigens is also presented.

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“…Calcium chelating substances (eg, EDTA) are sometimes more effective than citrate buffer in AR. 247,248,271,415 Calcium-induced changes in protein conformation may augment the immunodetection of some antigens, 399 but for many antigens, calcium-induced effects on immunoreactivity cannot be documented. 331,333 Restoring the native electrostatic charges modified during formalin fixation has also been considered an AR mechanism.…”

Section: Antigen Retrievalmentioning

confidence: 99%

When Tissue Antigens and Antibodies Get Along

2013

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Once focused mainly on the characterization of neoplasms, immunohistochemistry (IHC) today is used in the investigation of a broad range of disease processes with applications in diagnosis, prognostication, therapeutic decisions to tailor treatment to an individual patient, and investigations into the pathogenesis of disease. This review addresses the technical aspects of immunohistochemistry (and, to a lesser extent, immunocytochemistry) with attention to the antigen-antibody reaction, optimal fixation techniques, tissue processing considerations, antigen retrieval methods, detection systems, selection and use of an autostainer, standardization and validation of IHC tests, preparation of proper tissue and reagent controls, tissue microarrays and other high-throughput systems, quality assurance/quality control measures, interpretation of the IHC reaction, and reporting of results. It is now more important than ever, with these sophisticated applications, to standardize the entire IHC process from tissue collection through interpretation and reporting to minimize variability among laboratories and to facilitate quantification and interlaboratory comparison of IHC results.

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The Use of Monoclonal Antibodies and Limited Proteolysis in Elucidation of Structure—Function Relationships in Proteins

Cited by 50 publications

References 197 publications

Derepression of Bacterial Transcription-Repair Coupling Factor Is Associated with a Profound Conformational Change

Derepression of Bacterial Transcription-Repair Coupling Factor Is Associated with a Profound Conformational Change

Technical pitfalls potentially affecting diagnoses in immunohistochemistry

When Tissue Antigens and Antibodies Get Along

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