A modified Western blot protocol for enhanced sensitivity in the detection of a membrane protein

Kaur, Jaspreet; Bachhawat, Anand K.

doi:10.1016/j.ab.2008.10.005

Cited by 60 publications

(43 citation statements)

References 7 publications

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“…Total proteins (50 µg) were separated by SDS-PAGE (10% (w/v) polyacrylamide gel) and electroblotted (OmniPAGE Electroblotting Units, Cleaver Scientific Ltd) onto a nitrocellulose membrane (0.45 µm, Thermo Scientific). Membrane was then incubated in stripping buffer (10 mM Tris–HCl pH 6.8, 2% SDS and 100 mM β-mercaptoethanol) for 15 min at 55°C (as described by Kaur & Bachhawat [57]). Membrane was then treated with 5% non-fat dry milk, and immunodetection was done with a primary mouse anti-GFP monoclonal antibody (Roche Applied Science), or a mouse anti-actin monoclonal (C4) antibody (MP Biomedicals) and a secondary sheep anti-mouse IgG HRP-linked antibody (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

Modelling and mutational analysis of Aspergillus nidulans UreA, a member of the subfamily of urea/H ⁺ transporters in fungi and plants

et al. 2014

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We present the first account of the structure–function relationships of a protein of the subfamily of urea/H+ membrane transporters of fungi and plants, using Aspergillus nidulans UreA as a study model. Based on the crystal structures of the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT) and of the Nucleobase-Cation-Symport-1 benzylhydantoin transporter from Microbacterium liquefaciens (Mhp1), we constructed a three-dimensional model of UreA which, combined with site-directed and classical random mutagenesis, led to the identification of amino acids important for UreA function. Our approach allowed us to suggest roles for these residues in the binding, recognition and translocation of urea, and in the sorting of UreA to the membrane. Residues W82, Y106, A110, T133, N275, D286, Y388, Y437 and S446, located in transmembrane helixes 2, 3, 7 and 11, were found to be involved in the binding, recognition and/or translocation of urea and the sorting of UreA to the membrane. Y106, A110, T133 and Y437 seem to play a role in substrate selectivity, while S446 is necessary for proper sorting of UreA to the membrane. Other amino acids identified by random classical mutagenesis (G99, R141, A163, G168 and P639) may be important for the basic transporter's structure, its proper folding or its correct traffic to the membrane.

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Section: Methodsmentioning

confidence: 99%

Modelling and mutational analysis of Aspergillus nidulans UreA, a member of the subfamily of urea/H ⁺ transporters in fungi and plants

et al. 2014

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“…Proteins were separated on 12% or 7.5% gels and transferred to PVDF membranes as described above. Membranes to be probed with anti-TfR1 were then treated with 100 mM β-mercaptoethanol, 2% SDS in 62.5 mM tris buffer (pH 6.7) according to the protocol of Kaur and Bachhawat to enhance the signal of membrane-bound proteins (Kaur and Bachhawat, 2009). After washing, nonspecific sites of all membranes were blocked with 5% non-fat dry milk in a buffer containing 20 mM Tris, 500 mM NaCl, and 0.1% Tween 20 (pH 7.5) for 1 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Apotransferrin protects cortical neurons from hemoglobin toxicity

2011

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The protective effect of iron chelators in experimental models of intracerebral hemorrhage suggests that nonheme iron may contribute to injury to perihematomal cells. Therapy with high affinity iron chelators is limited by their toxicity, which may be due in part to sequestration of metals in an inaccessible complex. Transferrin is unique in chelating iron with very high affinity while delivering it to cells as needed via receptor-mediated endocytosis. However, its efficacy against iron-mediated neuronal injury has never been described, and was therefore evaluated in this study using an established cell culture model of hemoglobin neurotoxicity. At concentrations similar to that of CSF transferrin (50-100 micrograms/ml), both iron-saturated holotransferrin and apotransferrin were nontoxic per se. Overnight exposure to 3 μM purified human hemoglobin in serum-free culture medium resulted in death, as measured by lactate dehydrogenase release assay, of about three-quarters of neurons. Significant increases in culture iron, malondialdehyde, protein carbonyls, ferritin and heme oxygenase-1 were also observed. Holotransferrin had no effect on these parameters, but all were attenuated by 50-100 micrograms/ml apotransferrin. The effect of apotransferrin was very similar to that of deferoxamine at a concentration that provided equivalent iron binding capacity, and was not antagonized by concomitant treatment with holotransferrin. Transferrin receptor-1 expression was localized to neurons and was not altered by hemoglobin or transferrin treatment. These results suggest that apotransferrin may mitigate the neurotoxicity of hemoglobin after intracerebral hemorrhage. Increasing its concentration in perihematomal tissue may be beneficial.

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“…To increase the detection sensitivity for the transferred proteins, a modiWed Western blot protocol (Kaur and Bachhawat 2009) was used. After transfer of the proteins, the membrane was incubated in stripping buVer (62.5 mM Tris-HCl, pH 6.7, 100 mM -mercaptoethanol and 2% SDS) for 15 min at 55°C, followed by two washing steps with phosphate-buVered saline containing 0.05% Tween (PBST) at room temperature.…”

Section: Solubilization and Western Blot Analysis Of Membrane-bound Pmentioning

confidence: 99%

Mutagenesis and biochemical studies on AuaA confirmed the importance of the two conserved aspartate-rich motifs and suggested difference in the amino acids for substrate binding in membrane-bound prenyltransferases

Stec

2012

Arch Microbiol

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AuaA is a membrane-bound farnesyltransferase from the myxobacterium Stigmatella aurantiaca involved in the biosynthesis of aurachins. Like other known membrane-bound aromatic prenyltransferases, AuaA contains two conserved aspartate-rich motifs. Several amino acids in the first motif NXxxDxxxD were proposed to be responsible for prenyl diphosphate binding via metal ions like Mg(2+). Site-directed mutagenesis experiments demonstrated in this study that asparagine, but not the arginine residue in NRxxDxxxD, is important for the enzyme activity of AuaA, differing from the importance of NQ or ND residues in the NQxxDxxxD or NDxxDxxxD motifs observed in some membrane-bound prenyltransferases. The second motif of known membrane-bound prenyltransferases was proposed to be involved in the binding of their aromatic substrates. KDIxDxEGD, also found in AuaA, had been previously speculated to be characteristic for binding of flavonoids or homogenisate. Site-directed mutagenesis experiments with AuaA showed that KDIxDxEGD was critical for the enzyme activity. However, this motif is very likely not specific for flavonoid or homogenisate prenyltransferases, because none of the tested flavonoids was accepted by AuaA or its mutant R53A in the presence of farnesyl, geranyl or dimethylallyl diphosphate.

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A modified Western blot protocol for enhanced sensitivity in the detection of a membrane protein

Cited by 60 publications

References 7 publications

Modelling and mutational analysis of Aspergillus nidulans UreA, a member of the subfamily of urea/H ⁺ transporters in fungi and plants

Modelling and mutational analysis of Aspergillus nidulans UreA, a member of the subfamily of urea/H ⁺ transporters in fungi and plants

Apotransferrin protects cortical neurons from hemoglobin toxicity

Mutagenesis and biochemical studies on AuaA confirmed the importance of the two conserved aspartate-rich motifs and suggested difference in the amino acids for substrate binding in membrane-bound prenyltransferases

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A modified Western blot protocol for enhanced sensitivity in the detection of a membrane protein

Cited by 60 publications

References 7 publications

Modelling and mutational analysis of Aspergillus nidulans UreA, a member of the subfamily of urea/H + transporters in fungi and plants

Modelling and mutational analysis of Aspergillus nidulans UreA, a member of the subfamily of urea/H + transporters in fungi and plants

Apotransferrin protects cortical neurons from hemoglobin toxicity

Mutagenesis and biochemical studies on AuaA confirmed the importance of the two conserved aspartate-rich motifs and suggested difference in the amino acids for substrate binding in membrane-bound prenyltransferases

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Product

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About

Modelling and mutational analysis of Aspergillus nidulans UreA, a member of the subfamily of urea/H ⁺ transporters in fungi and plants

Modelling and mutational analysis of Aspergillus nidulans UreA, a member of the subfamily of urea/H ⁺ transporters in fungi and plants