Sean R. Gallagher

Immunoblotting and immunodetection.

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps starting with solubilization of the protein samples, usually with SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining or Ponceau S staining. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.

Staining proteins in gels.

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.

One-dimensional SDS gel electrophoresis of proteins.

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, that is, in the presence of sodium dodecyl sulfate (SDS). Both full‐size and minigel formats are detailed. Several modifications are provided for specific applications. For separation of peptides and small proteins, the standard buffers are replaced with either a Tris‐tricine buffer system or a modified Tris buffer in the absence of urea. Continuous SDS‐PAGE is a simplified method in which the same buffer is used for both the gel and electrode solutions and the stacking gel is omitted. Other protocols cover the preparation and use of ultrathin gels and gradient gels, and the simultaneous preparation of multiple gels.

Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy

Reliable quantitation of nanogram and microgram amounts of DNA and RNA in solution is essential to researchers using molecular biology techniques. In addition to the traditional absorbance measurements at 260 nm, two more sensitive fluorescence techniques are presented in this appendix. These three procedures cover a range from 5 to 10 ng DNA/ml to 50mg DNA/ml.

Immunoblotting and Immunodetection

This unit provides protocols for immunoblotting, which is used to identify specific protein sequences separated by electrophoresis and transferred to an appropriate membrane and recognized by a polyclonal or monoclonal antibody. After the proteins are separated on a gel, they are transferred to a membrane by electroblotting or a semidry transfer system. Proteins on the membrane can be visualized with the reversible stain Ponceau S to assess the completeness of transfer. Then the blot is analyzed with antibodies. The primary antibody is specific for the protein(s) of interest; the secondary antibody (an anti‐Ig) is conjugated to horseradish peroxidase or alkaline phosphatase and detected colorimetrically or by chemiluminescence. The membrane can be stripped and reused for other probes.

Detection of proteins on blot transfer membranes.

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.

APPENDIX 3D Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy

Quantitation of nucleic acids is a fundamental tool in molecular biology that requires accuracy, reliability, and the use of increasingly smaller sample volumes. This unit describes the traditional absorbance measurement at 260 nm and three more sensitive fluorescence techniques, as well as three microvolume methods that use fiber optic technology in specialized cells or instrumentation. These procedures allow quantitation of DNA solutions ranging from 1 pg/liter to 50 mg/ml.

UNIT 6.2 Immunoblotting and Immunodetection

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS‐PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody‐antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti‐IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed. Curr. Protoc. Cell Biol. 52:6.2.1‐6.2.28.

Quantitation of Nucleic Acids with Absorption Spectroscopy

This appendix describes a protocol for measuring absorption of nucleic acid samples at several different wavelengths to assess purity and concentration. A(260) measurements are quantitative for relatively pure nucleic acid preparations in microgram quantities. Absorbance readings cannot discriminate between DNA and RNA; however, the ratio of A at 260 and 280 nm can be used as an indicator of nucleic acid purity.

One‐Dimensional Electrophoresis Using Nondenaturing Conditions

Nondenaturing or “native” electrophoresis (i.e., electrophoresis in the absence of denaturants such as detergents and urea) is an often‐overlooked technique for determining the native size, subunit structure, and optimal separation of a protein. Because mobility depends on the size, shape, and intrinsic charge of the protein, nondenaturing electrophoresis provides a set of separation parameters distinctly different from mainly size‐dependent denaturing sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and charge‐dependent isoelectric focusing. Two protocols are presented in this unit. Continuous PAGE is highly flexible, permitting cationic and anionic electrophoresis over a full range of pH. The discontinuous procedure is limited to proteins negatively charged at neutral pH but provides high resolution for accurate size calibration.