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This unit describes protocols for detecting protein in a gel by Coomassie blue, silver, or fluorescent staining. As a general protein stain, Coomassie is easier and more rapid; however, fluorescent and silver staining methods are considerably more sensitive and thus can be used to detect smaller amounts of protein. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Alternate protocols describe rapid Coomassie and silver staining methods, as well as fluorescent stains that are specific for phosphoproteins and glycoproteins. Staining of proteins in SDS-polyacrylamide gels is described; variations for fluorescent staining of proteins in nondenaturing gels are also included. Support protocols describe photography of stained proteins.

Staining of blot transfer membranes permits visualization of proteins and allows the extent of transfer to be monitored. In the protocols described in this unit, proteins are stained after electroblotting from one-dimensional or two-dimensional polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. Protocols are provided for the use of six general protein stains: Amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, and India ink. In addition, the fluorescent stains fluorescamine and IAEDANS, which covalently react with bound proteins, are described. Approximate detection limits for each nonfluorescent stain are indicated along with membrane compatibilities.

The staining of proteins bound to a transfer membrane can be useful for determining the efficiency of transfer and marking the location of molecular weight standards. This unit describes three methods for staining blots, using India ink, gold labeling, and fluorescent labeling with SYPRO Ruby. Detection limits of each staining method are given along with a list of compatible blot transfer membranes and gels. A support protocol describes a method for enhancement of staining using alkali treatment., (Copyright 2008 by John Wiley & Sons, Inc.)

This unit describes protocols for detecting protein in a gel by either Coomassie blue, silver or fluorescent staining. Alternate rapid staining procedures are provided for each method and a support protocol describes how to photograph stained gels. Fluorescent staining (e.g., with SYPRO Orange or Red) is described as a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Staining of proteins in SDS-polyacrylamide gels is described, and an alternate protocol details variations in the procedure for proteins in nondenaturing gels. A final support protocol describes the photography of fluorescently stained proteins.

In the basic and alternate protocols of this unit, proteins are stained after electroblotting from polyacrylamide gels to blot transfer membranes. If the samples of interest are electrophoresed in duplicate and transferred to a blot transfer membrane, half of the membrane can be stained to determine the efficiency of transfer to the membrane and the other half can be used for immunoblotting (i.e., western blotting). Detection limits of each staining method are given along with a list of compatible blot transfer membranes and gels. A support protocol describes a method for alkali treatment that enhances subsequent staining of bound proteins.

This unit describes protocols for detecting protein in a gel by either Coomassie blue staining or silver staining. The former is easier and more rapid; however, silver staining methods are considerably more sensitive and thus can be used to detect smaller amounts of protein. Alternate rapid staining procedures are provided for each method and a support protocol describes how to photograph stained gels. Fluorescent staining is a popular alternative to traditional staining procedures, mainly because it is more sensitive than Coomassie staining, and often as sensitive as silver staining. Staining of proteins in SDS-polyacrylamide gels is described, and an alternate protocol details variations in the procedure for proteins in nondenaturing gels. A final support protocol describes the photography of fluorescently stained proteins.

A 22 x 10(3) Mr protein (abbreviated 22K) that copurifies with dermatan sulfate proteoglycans (DS-PGs) following the biochemical fractionation of bovine fetal skin has been evaluated for adhesion-promoting activity in vitro using Balb/c 3T3 cells, as well as bovine and human dermal fibroblasts. Substrata coated with 22K protein promote attachment of a subset of 3T3 and dermal fibroblasts that respond to plasma fibronectin (pFN) substrata. Cells on 22K protein display partial cytoplasmic spreading, comparable to that of cells adhering to cell-binding fragments of pFN. Adhesion activity of 22K is not due to contamination with known adhesive proteins of dermal matrices and is not dermal cell type-specific, since two classes of neuronal cells also respond effectively to 22K substrata. DS-PGs from cartilage or skin completely inhibit 22K adhesion activity when the PGs are adsorbed to 22K substrata under conditions prohibiting PGs from binding to substrata directly. Cartilage chondroitin/keratan sulfate proteoglycan at much higher concentrations is only partially inhibitory. Inhibition by DS-PGs is mediated by DS chains binding to 22K. Properties of the cell surface 'receptor' for 22K protein were tested by several approaches. It is not cell surface DS-PG, since: (1) cells unable to produce this proteoglycan class also responded; (2) cells treated with chondroitinase ABC responded equally well; and (3) substrata of proteoglycan-binding platelet factor-4 generated responses from cells that were quantitatively and qualitatively different. A synthetic peptide in the medium containing the Arg-Gly-Asp-Ser (RGDS) sequence completely inhibited responses to 22K substrata. This observation, coupled with sequencing data of 22K protein revealing an Arg-Gly-Ala-Thr sequence at residues 151-154, suggest that 22K protein mediates adhesion by cell surface integrin binding. Therefore, this newly discovered matrix protein from skin may serve as a communication link between the dermal fibroblast cell surface and its extracellular matrix environment.

Cartilage-derived growth factor purified to homogeneity by affinity chromatography on columns of heparin-Sepharose was mitogenic for early passage bovine fetal chondrocytes. Hyaluronate and sulfated glycosaminoglycan synthesis in these cells was analyzed by differential enzymatic digestion of the glycosaminoglycans labeled with [14C] glucosamine or [35S]. It was found that chondrocyte proliferation was accompanied by about a four-fold increase in hyaluronate synthesis over a two-day period, while the synthesis of sulfated glycosaminoglycans decreased by about 2-fold. Chromatographic analysis of the sulfated glycosaminoglycans showed decreases in chondroitin 4 and 6 sulfates. It was concluded from these results that cartilage-derived growth factor was a proliferative factor for chondrocytes and differed from the somatomedins.