Molecular and cell biological techniques

Methods

Molecular techniques
DNA-related RNA-related Protein-related

Cell biological methods

Methods

Molecular techniques

DNA and RNA-related

Southern blotting Northern blotting PCR RT-PCR Real-time PCR DNA/cDNA Microarray Subtractive hybridization SDS Polyacrylamide gel electrophoresis (SDS-PAGE) Western blotting Immunoprecipitation (pull down) Co-immunoprecipitation Far Western Two dimensional electrophoresis

Protein-related

Cell biological methods

Protein overexpression using cellular transfection/transduction RNA interference Immunocytochemistry FACS In situ hybridization Gene targeting Cell and animal transgenesis

Southern Blotting

Animation: Agarose gel Restriction

Pulse field gel electrophoresis

Northern Blotting

Polymerase chain reaction (PCR)

Animation: PCR PCR-1 PCR-Karp

Reverse Transcription-PCR (RT-PCR)

Animation: RT-PCR

Real time vs. conventional PCR

DNA/cDNA microarray

Animation: DNA microarray microarray microarray-karp

Experimental analysis of alternative splicing

Alternative splicing microarray

Matlin et al., Nat Rev Cell Mol Biol (2005)

Microarray profiling of alternative splicing

Blencow, Cell (2006)

SDS-PAGE

Immunoprecipitation

Animation: IP

Co-immunoprecipitation

Far Western

Two-dimensional electrophoresis

pH gradient set up first (using purchased mixture of ampholytes, different molecules designed to have range of pIs, which are first electrophoresed on the gel to form the pH gradient) Mixture of molecules (proteins) is then applied, electric field is turned on, and each protein moves to the position (pH) at which its net charge is zero, i.e., its pI.

Isoelectric focusing in first dimension, followed by SDS-PAGE at 90o to that (2nd dimension)

Protein over-expression

Animation: bacterial transformation transformation mechanism

Eukaryotic Expression Vectors

Immunocytochemistry

In most immunofluorescence experiments, two antibodies are employed. The first one, called the primary antibody, is typically generated in a mouse and binds to your favorite protein, which in this case is the chicken calcium ATPase (shown as a series undulating striped line that zigzags through teh ER membrane 10 times). The secondary antibody was purchased from a company that sells antibodies that bind to mouse antibodies and have a fluorescent dye covalently attached to it. As illustrated here, the secondary antibodies can bind to multiple sites on the primary antibody and thus produce a brighter signal since more dyes are brought to a single location.

Fluorescence Activated Cell Sorting (FACS)

In situ hybridization