James F. Battey

Western Blot Analysis

This chapter provides an overview of the western blot analysis method, which allows the investigator to identify specific proteins resolved by SDS polyacrylamide gel electrophoresis by binding with specific antisera. Proteins resolved on an acrylamide gel are transferred to a nitrocellulose (NC) filter that is incubated with the antisera. Transfer buffer used in the western blot analysis method consists of Tris base, glycine, water and methanol. The pH of the transfer buffer is maintained with HCl. The primary antibody specifically binds its epitope and the bound antibody is detected with a secondary species, such as [125] I]-protein A or biotinylated goat anti-IgG. The western blot analysis method involves the preparation of polyacrylamide–SDS gel for separation of proteins, and antibody is obtained against protein sequence of interest. The protein is electrophoretically transferred from the polyacrylamide gel to the NC filter.

Guanidine Isothiocyanate Preparation of Total RNA

This chapter discusses the method of guanidine isothiocyanate preparation of total RNA, which is a versatile and efficient way to extract intact RNA from most tissues and cultured cells, even if the endogenous level of RNase is high. The cells are lysed in guanidine isothiocyanate using a tissue homogenizer. The lysate is layered onto a CsCl gradient and spun in an ultracentrifuge. Proteins remain in the aqueous guanidine portion, DNA bands in the CsCl, and RNA pellets in the bottom of the tube. The RNA is recovered by redissolving the pellet. The recovery of RNA is usually excellent if the capacity of the gradient is not exceeded. The method can also be used to isolate RNA from tissue or to isolate both RNA and DNA from cells. The time taken to follow this method is two days.

Formaldehyde Gel for Electrophoretic Separation of RNA and Northern Blot

The formaldehyde/agarose gel is a simple denaturing electrophoresis system that allows good size separation and resolution of single-stranded RNA. The RNA is subsequently transferred to NC from the gel and hybridized with a radiolabeled probe of interest. The time required to carry out the procedure for electrophoretic separation of the RNA and northern blot is three days. On the first day, it takes five hours to run gel and set up the blot; on the second day, it takes three hours to bake NC filters after blotting and initiate hybridization; on the third day, it takes two hours to wash and set up autoradiography. This method requires a horizontal gel electrophoresis apparatus and fume hood. This chapter lists the reagents that are needed in this method, such as agarose, formaldehyde solution, ethidium bromide, and loading buffer

DNA Preparation from Cultured Cells and Tissue

This chapter explains DNA preparation from cultured cells and tissue. This method is used to prepare genomic DNA from tissue or cultured cells with excellent purity. It yields very high molecular weight DNA and involves an initial preparation of nuclei. This method is very time-consuming. On the first day, it takes 6–7 hours and on the second day, it takes 1–2 hours. The chapter further lists the reagents that are used in this process, such as diethyl ether, chloroform, isoamyl alcohol, ethanol, proteinase K, powdered form, TE buffer, and many more. Fresh tissue of interest for DNA isolation is prepared in advance. Blotting off blood and dissecting connective tissue are necessary in this method. Up to 5 g of tissue is used in this preparation. Alternatively, tissue can also be frozen and stored at -70°C. The chapter explains the preparation of tissue as well as nuclei.

Preparation of DNA from Eukaryotic Cells: General Method

This chapter explains the preparation of DNA from eukaryotic cells. This method provides a high-quality preparation of high molecular weight genomic DNA that is suitable for Southern blotting and library construction. Proteinase K is used to liberate nucleic acids from cells, followed by removal of RNA by RNase A. This method is used primarily with cultured cells. The chapter lists the reagents which are used in this process, such as phosphate-buffered saline (PBS), SS-phenol, chloroform, ethanol, TE buffer, and many more. Cells are grown for DNA extraction in advance. Tissue culture cells of various types work well. The chapter also explains DNA extraction.

Rapid DNA Preparation

This chapter discusses the method of rapid DNA preparation. This method allows the preparation of many small samples with a minimum of steps and time, generating DNA whose quality is adequate for Southern blot analysis of restriction fragments. Purified DNA is not good enough in quality for a DNA library because the Polytron shears the genomic DNA. The time required for rapid DNA preparation is 4–5 hours, and a polytron and shaker are required before carrying out the procedure. . The chapter lists the reagents that are used in this process, such as homogenization buffer, SS-phenol, chloroform, ethanol, and TE buffer.

Calcium Phosphate Transfection of Nonadherent and Adherent Cells with Purified Plasmids

Calcium phosphate transfection of nonadherent and adherent cells with purified plasmids is an efficient method for the introduction of cloned genes on recombinant plasmids into mammalian cells. There are slightly different protocols for use with nonadherent and adherent cells. For successfully carrying out the transfection procedure, cell culture equipment—such as incubator, biohood, and automatic pipettor—is required. All the cell lines are tested in advance to be used in transfection for sensitivity to glycerol. Preparation and purification of plasmid for transfection and sterilization of all buffers through 0.22-μm filters is done.

Selection of Poly(A + ) RNA on Oligo(dT) Cellulose

This chapter explains the method of selection of poly (A + ) RNA on oligo (dT) cellulose, which allows polyadenylated RNA species to be purified from nonpolyadenylated RNA. This step represents a considerable purification because mRNA represents only a small fraction of the total RNA in a eukaryotic cell. The method relies on base pairing between the poly (A + ) residues at the 3´ end of messenger RNAs and the oligo (dT) residues coupled to the cellulose column matrix. Nonpoly (A + ) species are not bound and are readily washed off the column. The bound poly (A + ) RNA is eluted by lowering the amount of salt in the column buffer. Poly (A + ) RNA gives better signals than total RNA in RNA gel blotting and Si nuclease protection experiments. It is also the starting material for cDNA library construction. The time required to follow this method is two hours. The equipment needed for this method is a spectrophotometer.

End Labeling of Synthetic Probes

This chapter discusses the end labeling of synthetic probes. The enzyme T4 polynucleotide kinase transfers the γ- 32 P label from ATP to a 5´ OH group of DNA or RNA. This reaction is used to end label DNA and RNA molecules such as oligonucleotides, restriction fragments, and mRNAs. The species labelled should have a 5´ OH group. Synthetic probes are typically supplied with a 5´ OH group. DNA restriction fragments and RNA have a 5´ phosphate terminus, requiring dephosphorylation with a phosphatase enzyme before labeling occurs. The chapter also presents a method for labeling on the 3´ end using the enzyme terminal deoxynucleotidyl transferase. The time required for the end labeling of synthetic probes is two hours. The chapter lists all the reagents that are needed for this method.

S 1 Nuclease Protection Assay

This chapter provides an overview of the S 1 nuclease protection assay. The assay allows the precise identification of pertinent gene boundaries in ribonucleic acid (RNA) transcripts. These can include intron/exon junctions and the 5´ or 3´ ends of transcripts. The S 1 nuclease protection assay allows a specific comparison between the RNA and a labeled DNA probe, which is a more stringent and sensitive method than the Northern blot analysis method but is also more tedious and requires more knowledge of the structure of the gene being expressed and analyzed. The Si nuclease assay works by the endonuclease digestion of single-stranded DNA. Thus, if the 32 P-DNA does not base pair precisely to the hybridizing RNA, extra tails or loops not protected by RNA hybridization will be excised. The chapter illustrates the use of denaturing polyacrylamide gel to determine the size of nondigested hybridized pieces. Resolution of the undigested S 1 -protected species yields detailed information about the regions of sequence homology between the probe and the protecting messenger RNA species.