Development of buffers for fast semidry transfer of proteins

Garić, Dušan; Humbert, Laure; Fils-Aimé, Nadège; Korah, Juliana; Zarfabian, Yasaman; Lebrun, Jean-Jacques; Ali, Suhad

doi:10.1016/j.ab.2013.07.009

Cited by 10 publications

(11 citation statements)

References 9 publications

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“…Proteins in gels were either stained with Coomassie brilliant blue G-250 (Diezel, Kopperschläger, & Hofmann, 1972) or transferred to a nitrocellulose membrane (BioTrace, Pall Corp.) using a fast semidry transfer buffer. (Garić et al, 2013) Polyclonal antibodies raised against the MBH large subunit (anti-HoxG, (Bernhard et al, 1996)) in combination with a goat anti-rabbit secondary antibody (coupled with alkaline phosphatase, Dianova) were used for detection of HoxG. Protein concentrations were determined with the Pierce BCA Protein Assay Kit (Thermo Scientific), using bovine serum albumin as standard.…”

Section: Protein Extract Preparation Polyacrylamide Gel Electrophoresis and Immunological Analysismentioning

confidence: 99%

A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation

et al. 2020

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[NiFe]‐hydrogenases catalyze the reversible conversion of molecular hydrogen into protons end electrons. This reaction takes place at a NiFe(CN)2(CO) cofactor located in the large subunit of the bipartite hydrogenase module. The corresponding apo‐protein carries usually a C‐terminal extension that is cleaved off by a specific endopeptidase as soon as the cofactor insertion has been accomplished by the maturation machinery. This process triggers complex formation with the small, electron‐transferring subunit of the hydrogenase module, revealing catalytically active enzyme. The role of the C‐terminal extension in cofactor insertion, however, remains elusive. We have addressed this problem by using genetic engineering to remove the entire C‐terminal extension from the apo‐form of the large subunit of the membrane‐bound [NiFe]‐hydrogenase (MBH) from Ralstonia eutropha. Unexpectedly, the MBH holoenzyme derived from this precleaved large subunit was targeted to the cytoplasmic membrane, conferred H2‐dependent growth of the host strain, and the purified protein showed exactly the same catalytic activity as native MBH. The only difference was a reduced hydrogenase content in the cytoplasmic membrane. These results suggest that in the case of the R. eutropha MBH, the C‐terminal extension is dispensable for cofactor insertion and seems to function only as a maturation facilitator.

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Section: Protein Extract Preparation Polyacrylamide Gel Electrophoresis and Immunological Analysismentioning

confidence: 99%

A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation

et al. 2020

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“…These issues are common and can lead to a decrease in the detection accuracy and reproducibility. Despite the improvements in the fixative reagents, electrode materials, transfer methods, and other steps in the western blotting process 8,10,11 , several challenges remain.…”

Section: Introductionmentioning

confidence: 99%

A fixation method for the optimisation of western blotting

Sun

Huang

et al. 2019

Sci Rep

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Western blotting is the most extensively used technique for the identification and characterisation of proteins and their expression levels. One of the major issues with this technique is the loss of proteins from the blotted membrane during the incubation and washing steps, which affects its sensitivity and reproducibility. Here, we have optimised the fixation conditions for immunoblotting and lectin blotting on electroblotted polyvinylidene difluoride and nitrocellulose membranes, using a combination of organic solvents and heating. Loss of proteins from polyvinylidene difluoride membranes was greatly reduced using this approach, the intensity of lectin blotting and immunoblotting was shown to increase 2.8- to 15-fold and 1.8- to 16-fold, respectively, compared with that samples without treated. Using the optimised method, cystic fibrosis transmembrane regulator and hypoxia-inducible factor 1, two difficult-to-analyse proteins with important physiological and pathological roles, were effectively detected. Additionally, it may help the identification of novel diagnostic markers for prostate cancer.

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“…20 µg of total protein was loaded on a gel and proteins were separated on a precast 4–15% polyacrylamide gradient gel (Bio-Rad, #4561085) by SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride (PVDF) membrane using semi-dry transfer and the fast semi-dry transfer buffer (1X, final) containing 48 mM Tris, 15 mM HEPPS with freshly added sodium bisulfate (1 mM final), EDTA (1.0 mM final) and 4 N, N-dimethylformamide (1.3 mM final) as previously described ( Garić et al, 2013 ). Membranes were incubated with primary antibodies: against the R domain of CFTR (23C5, provided by Dr. John Hanrahan lab) and against β-Actin (Santa Cruz, #sc-1616).…”

Section: Methodsmentioning

confidence: 99%

Treatment With LAU-7b Complements CFTR Modulator Therapy by Improving Lung Physiology and Normalizing Lipid Imbalance Associated With CF Lung Disease

et al. 2022

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Cystic fibrosis (CF) is the most common autosomal recessive genetic disease in Caucasians, affecting more than 100,000 individuals worldwide. It is caused by pathogenic variants in the gene encoding CFTR, an anion channel at the plasma membrane of epithelial and other cells. Many CF pathogenic variants disrupt the biosynthesis and trafficking of CFTR or reduce its ion channel function. The most frequent mutation, loss of a phenylalanine at position 508 (F508del), leads to misfolding, retention in the endoplasmic reticulum, and premature degradation of the protein. The therapeutics available for treating CF lung disease include antibiotics, mucolytics, bronchodilators, physiotherapy, and most recently CFTR modulators. To date, no cure for this life shortening disease has been found. Treatment with the Triple combination drug therapy, TRIKAFTA®, is composed of three drugs: Elexacaftor (VX-445), Tezacaftor (VX-661) and Ivacaftor (VX-770). This therapy, benefits persons with CF, improving their weight, lung function, energy levels (as defined by reduced fatigue), and overall quality of life. We examined the effect of combining LAU-7b oral treatment and Triple therapy combination on lung function in a F508deltm1EUR mouse model that displays lung abnormalities relevant to human CF. We assessed lung function, lung histopathology, protein oxidation, lipid oxidation, and fatty acid and lipid profiles in F508deltm1EUR mice.

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Development of buffers for fast semidry transfer of proteins

Cited by 10 publications

References 9 publications

A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation

A membrane‐bound [NiFe]‐hydrogenase large subunit precursor whose C‐terminal extension is not essential for cofactor incorporation but guarantees optimal maturation

A fixation method for the optimisation of western blotting

Treatment With LAU-7b Complements CFTR Modulator Therapy by Improving Lung Physiology and Normalizing Lipid Imbalance Associated With CF Lung Disease

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