The Bradford Method for Protein Quantitation

Hammond, John B. W.; Kruger, Nicholas J.

doi:10.1385/0-89603-126-8:25

Cited by 101 publications

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“…Protein content was determined by the dye-binding method of Bradford 4 as detailed by Hammond and Kruger 14 , using BSA as the standard and measuring the absorbance at 595 nm.…”

Section: Vsximr Qiewyviqirxmentioning

confidence: 99%

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8-11

Ogbonna¹,

Obi²,

Okolo³

et al. 2003

Journal of the Institute of Brewing

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A protease from sorghum malt variety KSV8-11 was purified by a combination of dialysis against 4 M sucrose, ion-exchange chromatography on Q-Sepharose (Fast flow), gel filtration chromatography on Sephadex G-100 and hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The enzyme was purified 5-fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg -1 protein. SDS-PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag + , Ca 2+ , Co 2+ , Fe 2+ , Mg 2+ , iodoacetic acid (IAA) and p-chloromercuribenzoate (p-CMB), stimulated by Cu 2+ , Sr 2+ , phenylmethylsulfonyl-fluoride (PMSF) and 2-mercaptoethanol (2-ME) while Mn 2+ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K m of 18 mg · mL -1 and a V max of 11.1 µmol · mL -1 · min -1 with casein as substrate.

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“…Protein content was determined by the dye-binding method of Bradford 4 as detailed by Hammond and Kruger 14 , using BSA as the standard and measuring the absorbance at 595 nm.…”

Section: Vsximr Qiewyviqirxmentioning

confidence: 99%

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8-11

Ogbonna¹,

Obi²,

Okolo³

et al. 2003

Journal of the Institute of Brewing

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“…After homogenization, the tissue homogenate was centrifuged at 600 × g for 5 min, and the supernatant was transferred to a new tube and centrifuged again at 3000 × g for 10 min. Protein concentration was quantified by the Bradford assay using BSA as the standard [21]. The total content of IL-10 (BD Bioscience, Franklin Lakes, NJ) and KIM-1 (R&D Systems, Minneapolis, MN) proteins in the kidney was quantified using specific ELISA kits.…”

Section: Methodsmentioning

confidence: 99%

Bone Marrow–Derived Mononuclear Cell Therapy Accelerates Renal Ischemia-Reperfusion Injury Recovery by Modulating Inflammatory, Antioxidant and Apoptotic Related Molecules

Ornellas¹,

Ornellas²,

Martini³

et al. 2017

Cell Physiol Biochem

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Background/Aims: We investigated the regenerative capacity of intravenous administration of bone marrow–derived mononuclear cells (BMMCs) in a rat model of bilateral renal ischemia/reperfusion (IR) injury and the involvement of inflammatory anti-inflammatory and other biological markers in this process. Methods: Rats were subjected to 1h bilateral renal pedicle clamping. BMMCs were injected i.v 1h after reperfusion and tracked by ^99mTc and GFP+ BMMCs. Twenty-four hours after reperfusion, renal function and histological changes were evaluated. The mRNA (real time PCR) and protein (ELISA and immuno-staining) expression of biological markers were analyzed. Results: Renal function and structure improved after infusion of BMMCs in the IR group (IR-C). Labeled BMMCs were found in the kidneys after therapy. The expression of inflammatory and biological markers (TLR-2, TRL-4, RAGE, IL-17, HMGB-1, KIM-1) were reduced and the expression of anti-inflammatory and antioxidant markers (IL-10, Nrf2, and HO-1) were increased in IR-C animals compared with IR untreated animals (IR-S). The apoptotic index diminished and the proliferation index increased in IR-C compared with IR-S. Conclusion: The results contribute to our understanding of the role of different biological players in morphofunctional renal improvement and cytoprotection in a post-ischemic reperfusion kidney injury model subjected to cellular therapy.

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“…The proteins were extracted from the cells using a passive lysis buffer (Promega Corporation, Madison, WI, USA) and their concentrations were determined using an assay, based on the Bradford method (Bio-Rad Laboratories, Inc., Hercules, CA, USA) as described previously (17). The protein samples (40 µg) were separated on 4-12% SDS-PAGE Ready Gels (Bio-Rad Laboratories, Inc.) and transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.).…”

Section: Methodsmentioning

confidence: 99%

Hepatitis C virus NS3 protein modulates the biological behaviors of malignant hepatocytes by altering the expression of host cell microRNA

Zhang

Ishigaki

Takegami

2015

Molecular Medicine Reports

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Abstract. Chronic hepatitis C virus (HCV) infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The HCV non-structural protein 3 (NS3) protein is considered to affect normal cellular functions and to be involved in HCV carcinogenesis. The expression of microRNA (miRNA) is altered in human HCC, thus implicating its role in hepatocarcinogenesis. To investigate the mechanisms by which the HCV NS3 protein affects the expression of miRNA in malignant hepatocytes, if any, the present study constructed expression vectors encoding the HCV NS3 and NS3/4A proteins, which were stably transfected into HepG2 cells. The biological behaviors of the HepG2 transfectants and their differential expression levels of miRNA expression were investigated. Compared with the HepG2-vector cells, the HepG2-NS3 cells grew at a slower rate, were arrested in the G0/G1 cell cycle phase, formed more colonies and developed larger tumors at a faster rate. Co-expression of HCV NS4A resulted in the inhibition of HCV NS3-stimulated tumorigenicity. A total of 35 miRNAs were dysregulated, 26 of which were downregulated and nine of which were upregulated, in the HepG2-NS3 cells, and 75 miRNAs were altered in HepG2-NS3/4A cells, of which 20 were downregulated and 55 were upregulated). In addition, significant decreases in the mRNA levels of p53 and p21 were observed, which confirmed differential expression of miRNA. These results suggested that differential miRNA profiling in malignant hepatocytes may account for the variable pathophysiological manifestations associated with the HCV NS3 protein. These differentially expressed miRNAs may offer potential as candidates for the development of miRNA-based therapeutics.

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The Bradford Method for Protein Quantitation

Cited by 101 publications

References 0 publications

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8-11

Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8-11

Bone Marrow–Derived Mononuclear Cell Therapy Accelerates Renal Ischemia-Reperfusion Injury Recovery by Modulating Inflammatory, Antioxidant and Apoptotic Related Molecules

Hepatitis C virus NS3 protein modulates the biological behaviors of malignant hepatocytes by altering the expression of host cell microRNA

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