Dunn M&C - Electrophoresis & Blotting

gel electrophoresis

a laboratory technique that separates molecules like DNA, RNA, and proteins by size and charge.

• the molecules are negatively charged and inserted into walls at one side of the gel (- side)

• smaller molecules move faster/further, larger ones move slowly

RNA/DNA gel electrophoresis

both migrate toward the positive electrode because of the negative charge in the phosphodiester backbone

DNA purification

1. cells are lysed using a detergent that disrupts the plasma membrane

2. protease and RNAase used to destroy protein and RNA

3. centrifuged, and a pellet is formed at the bottom. supernatant moved to a clean tube

4. DNA is precipitated w/ ethanol and forms viscous strands that can be spooled on a glass rod.

500-amino acid polypeptide length

172nm in length

gel electrophoresis process

biological molecules need to be uniformly negatively charged and be shaped like a noodle.

1. load cavities (“wells”) in gel with samples

2. hook up power supply and run gel. molecules separate over time as some migrate faster than others

3. remove gel after samples have run its length

process: formation of bands on gels

1. start with a mixture of molecules in a well

2. As electrophoresis starts, molecules begin to separate by size and charge

3. As electrophoresis continues, separation increases. Molecules with the same size and charge "run” at the same rate.

4. If each molecule is visualized, the result is a set of bands

DNA and RNA for electrophoresis

• DNA must be cut up (usually by restriction enzymes) before electrophoresis

• RNA must be denatured before (and during) electrophoresis

denaturing of molecules

disrupt weak non-covalent bonds but not covalent bonds (peptide bonds or phosphodiester bonds)

insulin

consists of two polypeptide that are bound by disulfide bonds

SDS-PAGE

electrophoresis of protein samples

• sodium dodecyl sulfate-polyacrylamide gel electrophoresis

housekeeping gene

one that virtually all cell types express.

they are often used as controls in experiments because their expression levels don’t vary too much from one cell type to another

blotting

transfer and virtually irreversible binding of biological molecules to a membrane (“magic paper”). uses probes/antibodies

can be used to detect specific electrophoresed DNA, RNA, or proteins

1. separate molecules by electrophoresis

2. transfer molecules to a paper-like membrane

3. detect specific molecule of interest with a detection molecule

detection of DNA/mRNA

(+) predictable rules for hybridization

(-) mRNA levels do not always correlate directly with protein levels (although they usually do)

• uses a DNA probe

DNA probe

using a labeled DNA probe to identify specific DNA sequences within a mixture of DNA fragments

• base pairs with what you’re trying to detect

detection of proteins

(+) antibodies allow one to measure the end product of protein-coding genes directly

(-) antibodies are less predictable than nucleic acids and more difficult to obtain

• di-sulfide bonds in proteins

• antibody

antibody

a protein produced by the immune system to recognize and neutralize foreign substances, like bacteria and viruses, called antigens

• used to detect specific protein bands on a blot after electrophoresis

blotting nucleic acids

steps in photo