Spectrophotometric and Colorimetric Determination of Protein Concentration

Simonian, Michael H.; Smith, John A.

doi:10.1002/0471142727.mb1001as76

Cited by 102 publications

(49 citation statements)

References 13 publications

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“…The stationary-phase parasites were harvested, washed in PBS (2 times), freeze-thawed (6 times), and centrifuged (16,000 ϫ g, 20 min, 4°C), and then the supernatant was collected, aliquoted, and stored in Ϫ70°C until use. The protein content of SLA was 500 g/ml as determined by the Bradford method (13).…”

Section: Methodsmentioning

confidence: 99%

Immune Responses of Iranian Patients with Visceral Leishmaniasis and Recovered Individuals to LCR1 of Leishmania infantum

Niknam

Abrishami

Doroudian

et al. 2014

Clin Vaccine Immunol

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Visceral leishmaniasis is a serious public health problem. Leishmania infantum is one of its causative agents. LCR1 is an immunogen from L. infantum. Antibodies against this protein have been detected in visceral leishmaniasis patients. The aim of this study was to define the antibody and cellular immune responses against LCR1 in Iranian visceral leishmaniasis patients and recovered individuals. The LCR1 protein was produced in recombinant form. Antibody responses against this protein were studied in Iranian individuals with a recent history of visceral leishmaniasis. Responses of peripheral blood mononuclear cells to this protein were studied in Iranian individuals who had recovered from visceral leishmaniasis. Our data show that (i) there was an antibody response to LCR1 in each individual with a recent history of visceral leishmaniasis studied, (ii) there was neither a proliferative response nor production of gamma interferon (IFN-␥) Leishmaniasis is a neglected disease resulting in a global mortality rate of approximately 60,000 per year. Visceral leishmaniasis (VL) is almost always fatal if left untreated. VL accounts for the majority of mortalities from leishmaniasis, represents a serious public health problem in regions where leishmaniasis is endemic, and is rapidly emerging as an opportunistic infection in HIV patients (1). Patients who recover from VL are resistant to reinfection (2). The fact that recovery from VL protects individuals from disease indicates that it should be possible to develop a vaccine against VL. However, there is no vaccine available for VL. There is great interest in finding immunogenic molecules from Leishmania infantum with potential applications as a vaccine candidate or a diagnostic molecule. Many immunogenic molecules from Leishmania infantum have been reported (1).Antibody responses are parts of the immune response against Leishmania infantum in humans. The presence of anti-Leishmania antibodies has been documented in VL (3, 4). The roles of these antibodies in immunity against VL in humans are not clear (2). However, the regulatory (5) and exacerbating (6) roles of antibodies in murine visceral leishmaniasis have been reported. AntiLeishmania antibodies have been used as diagnostic tools for this disease (7,8).Lymphocyte proliferation against Leishmania antigens is associated with protective immunity to VL (9). Gamma interferon (IFN-␥) is necessary for resistance against VL (10). Interleukin 10 (IL-10) is a counterprotective cytokine in VL (10).LCR1 is an immunogenic molecule discovered through screening a cDNA library of Leishmania infantum chagasi (11). This antigen has reacted with sera from Brazilian VL patients, showing the presence of anti-LCR1 in VL patients (11). Vaccination with LCR1 in a murine model of VL has shown some degree of protection against the disease (11). We were interested in whether LCR1 has potential uses as a vaccine or a diagnostic molecule in Iranian individuals. To approach these goals, we studied antibody responses against LCR1 in Iranian VL p...

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Section: Methodsmentioning

confidence: 99%

Immune Responses of Iranian Patients with Visceral Leishmaniasis and Recovered Individuals to LCR1 of Leishmania infantum

Niknam

Abrishami

Doroudian

et al. 2014

Clin Vaccine Immunol

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“…Human embryonic kidney (HEK) 293T cells (ATCC, cat. Additional reagents and equipment for determining protein concentration (Simonian and Smith, 2006) and protein precipitation (Support Protocol 4)…”

Section: Methodsmentioning

confidence: 99%

“…Centrifuge the cell lysates at 30 min at 21,000 × g, 4 • C, and combine the split fractions if necessary. Quantify the protein concentration using the Bradford assay (Simonian and Smith, 2006).…”

Section: Remove Supernatant and Resuspend Cell Pellet In 4 ML Tris-hcmentioning

confidence: 99%

Profiling Protein Methylation with Cofactor Analog Containing Terminal Alkyne Functionality

Blum

Bothwell

Islam

2013

CP Chemical Biology

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Enzymatic transmethylation from the cofactor S‐adenosyl‐L‐methionine (SAM) to biological molecules has recently garnered increased attention because of the diversity of possible substrates and implications in normal biology and diseases. To reveal the substrates of protein methyltransferases (PMTs), the present article focuses on an alkyne‐containing SAM mimic, Se‐adenosyl‐L‐selenomethionine (ProSeAM), and a cleavable azido‐azo‐biotin probe to profile the targets of endogenous PMTs in cellular contexts. This article describes the stepwise preparation of cell lysates containing active, endogenous PMTs and subsequent target labeling with ProSeAM. The article continues with the enrichment of the ProSeAM‐labeled proteins with the azido‐azo biotin probe as a pulldown reagent and the subsequent reductive elution with sodium dithionate for proteomic analysis. The protocols provided here were formulated for ProSeAM as a profiling reagent but can be applied to other terminal‐alkyne‐containing SAM analog cofactors. Curr. Protoc. Chem. Biol. 5:67‐88 © 2013 by John Wiley & Sons, Inc.

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“…The quantity of TSP was determined using the Bradford method [25] as previously described [26]. Bis-Tris 4-12% pre-cast gels (Life Technologies, Carlsbad, California, USA) were used for lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis (PAGE) under reducing conditions according to the manufacturer's protocol.…”

Section: Analysis Of Tobacco Hcps - Protein Quantification and 1-d Pagementioning

confidence: 99%

Polyclonal antibodies for specific detection of tobacco host cell proteins can be efficiently generated following RuBisCO depletion and the removal of endotoxins

Arfi

Hellwig

Drossard

et al. 2016

Biotechnology Journal

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The production of biopharmaceutical proteins in plants requires efficient downstream processing steps that remove impurities such as host cell proteins (HCPs) and adventitious endotoxins produced by bacteria during transient expression. We therefore strived to develop effective routines for endotoxin removal from plant extracts and the subsequent use of the extracts to generate antibodies detecting a broad set of HCPs. At first, we depleted the superabundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) for which PEG precipitation achieved the best results, preventing a dominant immune reaction against this protein. We found that a mixture of sera from rabbits immunized with pre-depleted or post-depleted extracts detected more HCPs than the individual sera used alone. We also developed a powerful endotoxin removal procedure using Polymyxin B for extracts from wild type plants or a combination of fiber-flow filtration and EndoTrap Blue for tobacco plants infiltrated with Agrobacterium tumefaciens. The antibodies we generated will be useful for quality and performance assessment in future process development and the methods we present can easily be transferred to other expression systems rendering them useful in the field of plant molecular farming.

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Spectrophotometric and Colorimetric Determination of Protein Concentration

Cited by 102 publications

References 13 publications

Immune Responses of Iranian Patients with Visceral Leishmaniasis and Recovered Individuals to LCR1 of Leishmania infantum

Immune Responses of Iranian Patients with Visceral Leishmaniasis and Recovered Individuals to LCR1 of Leishmania infantum

Profiling Protein Methylation with Cofactor Analog Containing Terminal Alkyne Functionality

Polyclonal antibodies for specific detection of tobacco host cell proteins can be efficiently generated following RuBisCO depletion and the removal of endotoxins

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