Native immunoblotting of blue native gels to identify conformation‐specific antibodies

Zickermann, Volker; Wumaier, Zibiernisha; Wrzesniewska, Blanka; Hunte, Carola

doi:10.1002/pmic.200900468

Cited by 5 publications

(2 citation statements)

References 16 publications

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“…The predicted position of subunit NUPM at the intermembrane space side of complex I could be confirmed by electron microscopy. The monoclonal antibody 31A8 of the IgG subtype had been shown to bind with high affinity to complex I from Y. lipolytica in native and denaturing ELISA variants [46]. However, it failed to recognize any complex I polypeptide in a standard denaturing dSDS-PAGE/Western blot analysis.…”

Section: Resultsmentioning

confidence: 99%

A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I

Angerer

Zwicker

Wumaier

et al. 2011

Biochemical Journal

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Synopsis Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a very large membrane protein complex with a central function in energy metabolism. Complex I from the aerobic yeast Yarrowia lipolytica comprises 14 central subunits that harbour the bioenergetic core functions and at least 28 accessory subunits. Despite progress in structure determination the position of individual accessory subunits in the enzyme complex remains largely unknown. Proteomic analysis of subcomplex Iδ revealed that it lacked eleven subunits including the central subunits ND1 and ND3 forming the interface between the peripheral and the membrane arm in bacterial complex I. This unexpected observation provided insight into the structural organization of the connection between the two major parts of mitochondrial complex I. Combining recent structural information, biochemical evidence on the assignment of individual subunits to the subdomains of complex I and sequence based predictions for the targeting of subunits to different mitochondrial compartments, we derived a model for the arrangement of the subunits in the membrane arm of mitochondrial complex I.

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Section: Resultsmentioning

confidence: 99%

A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I

Angerer

Zwicker

Wumaier

et al. 2011

Biochemical Journal

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“…Native PAGE might be considered as a compromise . Notably, native PAGE and native protein transfer could be easily combined with our lane cutting approach.…”

Section: Discussionmentioning

confidence: 99%

Completing the Immunological Fingerprint by Refractory Proteins: Autoantibody Screening via an Improved Immunoblotting Technique

Moritz

Tholance

Rosier

et al. 2019

Proteomics Clinical Apps

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Purpose: Identifying autoantigens of serological autoantibodies requires expensive methods, such as protein microarrays or IP+MS. Thus, sera are commonly pre-screened for interesting immunopatterns via immunocytochemistry/immunohistochemistry. However, distinguishing immunopatterns can be difficult and intracellular antigens are less accessible. Therefore, a simple and cheap immunoblot screening able to distinguish immunopatterns and to detect refractory proteins is presented. Experimental Design: Five steps of immunoblotting-based autoantigen screening are revised: (1) choice of protein source, (2) protein extraction, (3) protein separation, (4) protein transfer, (5) antigen detection. Thereafter, 52 patients' sera with chronic inflammatory demyelinating polyneuropathy (CIDP) and 45 controls were screened. Results: The protein source impacts the detected antigen set. Steps 2-4 can be adapted for refractory proteins. Furthermore, longitudinal cutting of protein lanes saves ࣙ75% of time and material and allows for exact comparison of band patterns. As the latter are individually specific and temporarily constant, we call them "immunological fingerprints". In a proof-of-principle, a 155 kDa immunoband was detected with two anti-neurofascin-155-positive CIDP sera and two further immunobands (120/220 kDa) specific to a subgroup of 3-6 of 52 CIDP patients. Conclusions and Clinical Relevance: Adapted immunoblotting is a cheap and simple method for accurate serum screening including refractory and intracellular antigens.

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Native electrophoresis in cell proteomic: BN-PAGE and CN-PAGE

2011

Cell Tiss. Biol.

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Native immunoblotting of blue native gels to identify conformation‐specific antibodies

Cited by 5 publications

References 16 publications

A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I

A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I

Completing the Immunological Fingerprint by Refractory Proteins: Autoantibody Screening via an Improved Immunoblotting Technique

Native electrophoresis in cell proteomic: BN-PAGE and CN-PAGE

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