Chapter 33 Performing and Optimizing Western Blots with an Emphasis on Chemiluminescent Detection

Alegria-Schaffer, Alice; Lodge, Andrew J.; Vattem, Krishna M.

doi:10.1016/s0076-6879(09)63033-0

Cited by 122 publications

(90 citation statements)

References 16 publications

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“…Western blot band number 4 after 15 min digestion appears as a so-called "ghost band" likely due to sample or detection antibody overloading as previously described. 42 A »38 kDa band observed in the untreated IFNAR1 lane of the western blot was not detectable in the SDS-PAGE gel. This band is likely a minor degradation product that pre-existed in the initial protein preparation and was digested into smaller fragments upon endoproteinase treatment.…”

Section: Determination Of Anifrolumab Epitope Using Deletion Variantsmentioning

confidence: 88%

Molecular basis for antagonistic activity of anifrolumab, an anti-interferon–α receptor 1 antibody

Li¹,

Oganesyan²,

Wu³

et al. 2015

mAbs

104

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Anifrolumab (anifrolumab) is an antagonist human monoclonal antibody that targets interferon a receptor 1 (IFNAR1). Anifrolumab has been developed to treat autoimmune diseases and is currently in clinical trials. To decipher the molecular basis of its mechanism of action, we engaged in multiple epitope mapping approaches to determine how it interacts with IFNAR1 and antagonizes the receptor. We identified the epitope of anifrolumab using enzymatic fragmentation, phage-peptide library panning and mutagenesis approaches. Our studies revealed that anifrolumab recognizes the SD3 subdomain of IFNAR1 with the critical residue R 279 . Further, we solved the crystal structure of anifrolumab Fab to a resolution of 2.3 A . Guided by our epitope mapping studies, we then used in silico protein docking of the anifrolumab Fab crystal structure to IFNAR1 and characterized the corresponding mode of binding. We find that anifrolumab sterically inhibits the binding of IFN ligands to IFNAR1, thus blocking the formation of the ternary IFN/ IFNAR1/IFNAR2 signaling complex. This report provides the molecular basis for the mechanism of action of anifrolumab and may provide insights toward designing antibody therapies against IFNAR1.

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Section: Determination Of Anifrolumab Epitope Using Deletion Variantsmentioning

confidence: 88%

Molecular basis for antagonistic activity of anifrolumab, an anti-interferon–α receptor 1 antibody

Li¹,

Oganesyan²,

Wu³

et al. 2015

mAbs

104

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“…Standard method was followed as previously described (1). GFRP (Proteintech), GTPCH1 (Santa Cruz Biotechnology, Inc.), and b-actin (Cell Signaling Technology) was used at 1:1000 ratio, while secondary antibody was used at a ratio of 1:20,000.…”

Section: Western Blotting Analysismentioning

confidence: 99%

Characterization of TransgenicGfrpKnock-In Mice: Implications for Tetrahydrobiopterin in Modulation of Normal Tissue Radiation Responses

Pathak

Pawar

et al. 2014

Antioxidants & Redox Signaling

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Aims: The free radical scavenger and nitric oxide synthase cofactor, 5,6,7,, plays a well-documented role in many disorders associated with oxidative stress, including normal tissue radiation responses. Radiation exposure is associated with decreased BH4 levels, while BH4 supplementation attenuates aspects of radiation toxicity. The endogenous synthesis of BH4 is catalyzed by the enzyme guanosine triphosphate cyclohydrolase I (GTPCH1), which is regulated by the inhibitory GTP cyclohydrolase I feedback regulatory protein (GFRP). We here report and characterize a novel, Cre-Lox-driven, transgenic mouse model that overexpresses Gfrp. Results: Compared to control littermates, transgenic mice exhibited high transgene copy numbers, increased Gfrp mRNA and GFRP expression, enhanced GFRP-GTPCH1 interaction, reduced BH4 levels, and low glutathione (GSH) levels and differential mitochondrial bioenergetic profiles. After exposure to total body irradiation, transgenic mice showed decreased BH4/7,8-dihydrobiopterin ratios, increased vascular oxidative stress, and reduced white blood cell counts compared with controls. Innovation and Conclusion: This novel Gfrp knock-in transgenic mouse model allows elucidation of the role of GFRP in the regulation of BH4 biosynthesis. This model is a valuable tool to study the involvement of BH4 in whole body and tissue-specific radiation responses and other conditions associated with oxidative stress.

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“…a linear correlation between protein amount and analytical signal, is mostly unknown when quantifying proteins by Western blot using chemiluminescent signals for detection (76,79). Associated to this, we could demonstrate a highly variable but linear correlation for the water-soluble protein HSA but rather a quadratic correlation for hepatic OATP transporters (78).…”

Section: Protein Quantification By Immunoblottingmentioning

confidence: 67%

“…cross-reactivity with other proteins or even a lack of functionality (76,77). This is not surprising when one keeps in mind that an epitope which is recognized by specific antibodies has normally the length of five to ten amino acids and its distinct binding motif is mostly unknown.…”

Section: Protein Quantification By Immunoblottingmentioning

confidence: 99%

Mass Spectrometry-Based Targeted Proteomics as a Tool to Elucidate the Expression and Function of Intestinal Drug Transporters

Oswald

Gröer

Droździk

et al. 2013

AAPS J

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Abstract. Intestinal transporter proteins affect the oral bioavailability of many drugs in a significant manner. In order to estimate or predict their impact on oral drug absorption, data on their intestinal expression levels are needed. So far, predominantly mRNA expression data are available which are not necessarily correlated with the respective protein content. All available protein data were assessed by immunoblotting techniques such as Western blotting which both possess a number of limitations for reliable protein quantification. In contrast to this, mass spectrometry-based targeted proteomics may represent a promising alternative method to provide comprehensive protein expression data. In this review, we will summarize so far available intestinal mRNA and protein expression data for relevant human multidrug transporters. Moreover, recently observed mass spectrometry-based targeted proteomic data will be presented and discussed with respect to potential functional consequences. Associated to this, we will provide a short tutorial how to set up these methods and emphasize critical aspects in method development. Finally, potential limitations and pitfalls of this emerging technique will be discussed. From our perspective, LC-MS/MS-based targeted proteomics represents a valuable new method to comprehensively analyse the intestinal expression of transporter proteins. The resulting expression data are expected to improve our understanding about the intestinal processing of drugs.

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Chapter 33 Performing and Optimizing Western Blots with an Emphasis on Chemiluminescent Detection

Cited by 122 publications

References 16 publications

Molecular basis for antagonistic activity of anifrolumab, an anti-interferon–α receptor 1 antibody

Molecular basis for antagonistic activity of anifrolumab, an anti-interferon–α receptor 1 antibody

Characterization of TransgenicGfrpKnock-In Mice: Implications for Tetrahydrobiopterin in Modulation of Normal Tissue Radiation Responses

Mass Spectrometry-Based Targeted Proteomics as a Tool to Elucidate the Expression and Function of Intestinal Drug Transporters

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