Optimization of western blotting for the detection of proteins of different molecular weight

Protein samples electroblotted onto nitrocellulose membranes and quenched with a mixture of blocking agents produced a strong signal for cystic fibrosis transmembrane-conductance regulator (CFTR), a high-molecular-weight protein, in western blotting. Optimized conditions for CFTR were then extended to medium- and low-molecular-weight proteins (LAMP1 and Rab11a, respectively) to determine the effects of methanol concentration (0–20%) in Towbin’s transfer buffer (TTB). Methanol in TTB appears to have little to no effect on CFTR signal. However, for medium-sized (LAMP1) and small (Rab11a) proteins, a lower concentration of methanol (10%) was sufficient to produce a maximal signal. Therefore, methanol, a toxic solvent, can be removed from or reduced in TTB without compromising signal strength. Here, we show modifications that may be useful in detecting and/or improving the signal of low-abundance proteins.

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Western Blotting of High and Low Molecular Weight Proteins Using Heat

Methods in Molecular Biology - Western Blotting ◽

10.1007/978-1-4939-2694-7_26 ◽

2015 ◽

pp. 247-255 ◽

Cited By ~ 3

Author(s):

Biji T. Kurien ◽

R. Hal Scofield

Keyword(s):

Molecular Weight ◽

Western Blotting ◽

Low Molecular Weight ◽

Low Molecular Weight Proteins

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Urinary Albumin and Low Molecular Weight Protein Excretion in the Nephrotic Syndrome—Sequential Studies during Corticosteroid Treatment

Annals of Clinical Biochemistry International Journal of Laboratory Medicine ◽

10.1177/000456329202900414 ◽

1992 ◽

Vol 29 (4) ◽

pp. 450-453 ◽

Cited By ~ 4

Author(s):

Robert Beetham ◽

David Newman

Keyword(s):

Molecular Weight ◽

Nephrotic Syndrome ◽

Preliminary Investigation ◽

Corticosteroid Treatment ◽

Retinol Binding Protein ◽

Low Molecular Weight ◽

Proximal Tubular ◽

Weight Protein ◽

Tubular Absorption ◽

Low Molecular Weight Proteins

In a preliminary investigation into the behaviour of low molecular weight proteins in the nephrotic syndrome, we have measured urinary concentrations of albumin, α-1-microglobulin (α1-m) and retinol-binding protein (RBP) in six children for up to 11 days during the course of steroid therapy for nephrotic syndrome. The results in part support the concept of independent proximal tubular absorption of albumin and low molecular weight proteins, and indicate that in the nephrotic syndrome the excretion of RBP and α1-m, two generally accepted markers of tubular proteinuria, is anomalous.

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Breakdown of mucin and plant polysaccharides in the human colon

Canadian Journal of Biochemistry ◽

10.1139/o77-178 ◽

1977 ◽

Vol 55 (11) ◽

pp. 1190-1196 ◽

Cited By ~ 46

Author(s):

J. R. Vercellotti ◽

A. A. Salyers ◽

W. S. Bullard ◽

T. D. Wilkins

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Human Subjects ◽

Human Colon ◽

Low Molecular Weight ◽

High Molecular Weight Protein ◽

Complex Carbohydrate ◽

Plant Polysaccharides ◽

Weight Protein ◽

Molecular Weight Fractions

To obtain an estimate of the extent to which complex carbohydrates are degraded by bacteria in the human colon, aqueous extracts of colon contents from four human subjects were separated into high and low molecular weight fractions by chromatography on a Sephadex G-100 column. The composition of these fractions was compared with the composition of similar fractions from ileal contents, i.e., from material entering the colon. In all four subjects, high molecular weight carbohydrate concentrations were lower in the colon than in the ileum, indicating that breakdown of complex carbohydrate occurs in the colon. The high molecular weight carbohydrate fraction contained sugars characteristic of plant polysaccharides (arabinose, xylose, mannose, rhamnose) as well as sugars characteristic of mucin (fucose, hexosamines, sialic acids). Concentrations of most of these sugars were uniformly lower in the colon than in the ileum. Since high molecular weight protein concentrations were lower in the colon than in the ileum of two of the four subjects tested, some degradation of protein may also occur in the colon.

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Protein kinase C and Ca2+ activation of mucin secretion in airway goblet cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1997.273.1.l201 ◽

1997 ◽

Vol 273 (1) ◽

pp. L201-L210 ◽

Cited By ~ 15

Author(s):

L. H. Abdullah ◽

J. D. Conway ◽

J. A. Cohn ◽

C. W. Davis

Keyword(s):

Cystic Fibrosis ◽

Protein Kinase C ◽

Protein Kinase ◽

Western Blotting ◽

Goblet Cells ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Mucin Secretion ◽

Kinase C ◽

Cystic Fibrosis Transmembrane

Airway goblet cells secrete mucin in response to ATP and uridine 5'-triphosphate (UTP), but the underlying signal transduction pathways are poorly understood. Cultures of SPOC1 cells (L. H. Abdullah, S. W. Davis, L. Burch, M. Yamauchi, S. H. Randell, P. Nettesheim, and C. W. Davis. Biochem. J. 316: 943-951, 1996) secreted mucin on exposure to phorbol 12-myristate 13-acetate (PMA) [apparent affinity (K0.5) approximately 100 nM] and ionomycin (K0.5 approximately 5 microM) almost fivefold over baseline. Thapsigargin also elicited secretion (K0.5 approximately 20 nM). Ionomycin and PMA together elicited approximately twice the secretion of either agent alone. Overnight exposure to half-maximal PMA abolished the response to maximal doses of UTP and PMA, whereas ionomycin was fully effective. Protein kinase C (PKC) activity in the membrane fraction was increased by maximal doses of PMA and UTP, whereas ionomycin had no effect. PKC inhibitors were relatively ineffective against PMA- and UTP-induced mucin secretion. Human and canine goblet cells in epithelial explants, by video microscopy, underwent exocytosis with ionomycin (1 microM) and PMA (0.1 or 1 microM). SPOC1 cell mucin secretion was not stimulated by forskolin, 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate, or 8-bromoguanosine 3',5'-cyclic monophosphate. Cystic fibrosis transmembrane conductance regulator was not detected in SPOC1 cells by Western blotting, and its mRNA was detected by reverse transcriptase polymerase chain reaction (PCR) only as a very weak band and after 55 PCR cycles. Multidrug resistance (MDR1), however, was readily detected by Western blotting, and its mRNA was detected as a major band after 35 PCR cycles. Thus airway goblet cell mucin secretion, distal to receptor activation, may be regulated independently by Ca(2+)- and PKC-dependent pathways. Cystic fibrosis transmembrane conductance regulator and cyclic nucleotides, however, may not play a major role in this secretion.

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