Proteins general protocol

The following is a generalized protocol for the activation of a protein with sulfo-SMCC with subsequent conjugation to a sulfhydryl-containing second molecule or protein. Specific examples of the use of this crosslinker to make antibody-enzyme or hapten-carrier conjugates may be found in Chapter 20, Section 1.1 and Chapter 19, Section 5, respectively. 

A general protocol for the modification of proteins, particularly immunoglobulins, with FITC is given below. Slight modifications to the amount of reagent added to the reaction may be done to optimize the F/P ratio. 

The following generalized protocol relates to the labeling of IgG with NHS-rhodamine. Optimization of the level of rhodamine incorporation may have to be done with other proteins or other macromolecules. 

A general protocol for the use of DCIA for fluorescently labeling proteins that contain sulfhydryl residues may be obtained by following the method discussed for AMCA-HPDP (previous section). After purification of the labeled protein, the F/P ratio of fluorophore incorporation may be determined by measuring its 382nm/280nm absorbance ratio. 

Dissolve the protein or antibody to be conjugated in 0.1 M sodium phosphate, 0.15M NaCl, pH 7.4. The antibody solution should be as concentrated as possible, given the amount of antibody available for modification. This general protocol will work for protein or antibody concentrations ranging from about 0.5mg/ml to lOmg/ml, but an increase in the molar excess of either SANH or SFB may have to be done at lower antibody concentrations to provide the same modification yields obtained at higher concentrations. 

A general protocol that can serve as a guide for the use of heterobifunctional crosslinkers in the study of protein interactions is given below. Some optimization of concentrations may have to be done depending on the particular type and properties of proteins being studied. 

The Search for a General Protocol for Protein Solution Synthesis... 

Avidin conjugates of a wide range of fluorophores, phycobiliprotems, secondary antibodies, microspheres, ferritin, and enzymes commonly used in lmmunochemistry are available at reasonable prices, making their small scale preparation impractical and not cost effective (see Note 1). However, conjugations of avidin to specific antibodies, to uncommon enzymes, and to other proteins and peptides are often performed on-site. A general protocol for the conjugation of avidin to enzymes, antibodies, and other proteins is described in this chapter. 

Generalized protocols for the attachment of these fluorophores to protein molecules, including antibodies, can be found in Chapter 8, Section 1. The main consider-... 

The general protocol for intracellular staining involves, first, staining the cells for any surface (outer membrane) antigens, as described in the previous chapter. Then the surface proteins with their... 

The theory behind such techniques is complex and not clearly understood, but the techniques themselves are straightforward. To say that an increase in protein-protein interactions leads to precipitation whereas protein-solvent interaction favors solubility is a simplistic but nonetheless useful paradigm. Precipitation techniques do not, by themselves, achieve a great increase in the purity of a protein solution, but generally they result in an increase in concentration and have a role to play in many protein purification protocols. 

Western blotting is one of the most widely used immunochemical techniques in modern biochemistry. In general, it is used to detect the presence of a particular protein antigen in an impure mixture. Western blots can provide information that is (1) qualitative Is the protein present (2) quantitative How much of the protein is present and (3) structural What is the size of the protein The general protocol for Western blotting is as follows (see Fig. 18-1) ... 

We prepare total brain protein extract according to a general protocol (Fountou-lakis et al., 2002). Brain tissue (ca. 0.25 g) is placed in BiolOl tubes (fast RNA tubes). To each tube, 0.8 mb of 20 mM Tris, which contains 7 M urea, 2 M thiourea,... 

Table I. The general protocol for collection of bulk and adsorbed protein spectra...

A general protocol for mass spectrometric analysis of phosphoproteins is illustrated in Figure 15 various steps of this protocol are cleavage of purified phosphoproteins, isolation and preferential enrichment of phosphopeptides, selective detection of phosphopeptides in the digest, identification of the phosphorylation sites using tandem mass spectrometry, and identification of phosphopeptides/proteins through a database search. 

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