Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes

Moeremans, M.; Daneels, Guy; Mey, J. De

doi:10.1016/0003-2697(85)90368-9

Cited by 235 publications

(96 citation statements)

References 15 publications

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“…Other high sensitivity protocols involve the use of colloidal metal particles. AuroDye ® staining [115,116] is based on the hydrophobic and ionic interaction between colloidal gold particles and proteins at pH 3; dark red bands/spots develop. Sensitivity is equal or higher than with silver stain in gels [117], and 1-100 ng protein can be quantified by scanning densitometry [116,118].…”

Section: Non-reversible Proceduresmentioning

confidence: 99%

Protein stains for proteomic applications:Which, when, why?

Crawford²,

2006

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This review recollects literature data on sensitivity and dynamic range for the most commonly used colorimetric and fluorescent dyes for general protein staining, and summarizes procedures for the most common PTM-specific detection methods. It also compiles some important points to be considered in imaging and evaluation. In addition to theoretical considerations, examples are provided to illustrate differential staining of specific proteins with different detection methods. This includes a large body of original data on the comparative evaluation of several pre-and post-electrophoresis stains used in parallel on a single specimen, horse serum run in 2-DE (IPG-DALT). A number of proteins/protein spots are found to be over-or under-revealed with some of the staining procedures.

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Section: Non-reversible Proceduresmentioning

confidence: 99%

Protein stains for proteomic applications:Which, when, why?

Crawford²,

2006

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“…Membranes were blocked with TBS containing 0.1% (v/v) Tw for 30 min, cut into strips, incubated with Ab dilution, and developed by the alkaline phosphatase method (23). Strips were stained with colloidal gold to visualize the proteins on the blot (24). For estimating the apparent molecular size of mMBL by gel-permeation chromatography of mouse serum, the Western blots of the fractions were incubated with biotinylated second Ab and developed by enhanced chemiluminescence using HRP-labeled streptavidin (Dako) at 0.2 g/ml and luminescence reagent (Pierce).…”

Section: Western Blottingmentioning

confidence: 99%

Purification and Characterization of Two Mannan-Binding Lectins from Mouse Serum

Hansen

Thiel

Willis

et al. 2000

The Journal of Immunology

100

107

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Mannan-binding lectin (MBL) is a serum protein that activates the complement system after binding to glycoconjugates found on the surface of microorganisms. By molecular cloning two forms of MBL have been identified in the mouse (mMBL-A and mMBL-C), but only mMBL-A has been purified and characterized at the protein level. MBL-C has been termed the liver form of MBL. The present report describes the purification and characterization of mMBL-A and mMBL-C from serum. The two forms of mMBL could be separated both by ion-exchange and carbohydrate-affinity chromatography. The initial identification by immunochemical technique was confirmed by N-terminal amino-acid sequencing. Both proteins give bands corresponding to polypeptide chains of 28 kDa on SDS-PAGE in the reduced state, but mMBL-A migrated more rapidly than mMBL-C in acid/urea-PAGE, in accordance with the calculated pIs. Both forms mediated activation of complement component C4 in mannan-coated microtiter wells. MBL-A showed a higher affinity for d-glucose and α-methyl-d-glucose then did MBL-C. Serum concentrations of mMBL-A in laboratory strains and wild mice were found to vary from 5 to 80 μg/ml, with wild mice tending to show higher levels than laboratory strains.

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“…In our laboratory, we found that the established assays consumed significant quantities of valuable sample preparations. As we were using colloidal gold to stain proteins on nitrocellulose Western blots, we investigated the possibility that the same reaction could be used to quantitatively estimate protein concentration [7]. We developed a protein assay in which a small volume (2 μl) of protein solution is applied to nitrocellulose paper in a grid array, and dried.…”

Section: Introductionmentioning

confidence: 99%

A nanogram-level colloidal gold single reagent quantitative protein assay

Harrison

Haffey

Golub

2008

Analytical Biochemistry

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We have developed a nanogram level quantitative protein assay based on the binding of colloidal gold to proteins adhered to nitrocellulose paper. The protein-gold complex produces a purple color proportional to the amount of protein present, and the intensity of the stain is quantified by densitometry. Typical assays require minimal starting material (10 -20 μl) containing 2 -5 μg protein. A small volume (2 μl) of protein solution is applied to nitrocellulose paper in a grid array, and dried. The nitrocellulose is incubated in colloidal gold suspension with gentle agitation (4-16 h), rinsed with water and scanned. Densitometric analysis of the scanned images allows quantitation of the unknown sample protein concentration by comparison with protein standards placed on the same nitrocellulose grid. The assay requires significantly less sample than conventional protein assays. In this report, the Golddots assay is calibrated against weighed protein samples, and compared with the Pierce Micro BCA protein assay kit. In addition, the assay is evaluated with several known proteins with different physical properties.

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Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes

Cited by 235 publications

References 15 publications

Protein stains for proteomic applications:Which, when, why?

Protein stains for proteomic applications:Which, when, why?

Purification and Characterization of Two Mannan-Binding Lectins from Mouse Serum

A nanogram-level colloidal gold single reagent quantitative protein assay

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