Mol Bio Techniques

Light Microscopy

Uses wavelength to pick up Prokaryotic Cells and Eukaryotic Cell structures

Fluorescent Microscopy

Uses Flourophores that Quantum jumps to reflect a lower energy photon after exposed to light. It gives rough locations of protein/structures

FRET

It uses two Flourophores, one donor that donates photon to the receptor fluorophore. If the Acceptor is near the donor, it will send a lower energy photon back to the donor and it’s reflected. It is used to look at if there is INTERACTIONS between proteins or components of a large protein.

Cryo-EM

Uses electron particle-wave duality to shoot an electron beam at a frozen solution with protein. The light produces a bunch of shadows that are pieced together. It is used to determine secondary and tertiary structures of proteins or macromolecules.

X-Ray Crystallography

Crystallize proteins all in same orientation. Shines a x-ray beam through it and the diffractions determine protein structure. it is used to determine Primary and Secondary Protein structures.

NMR

Odd isotopes have a funky spin and when you use a radio wave to bounce it’s electron off a magnet, it will release a signal when getting back on the magnet. It is used to look at the dynamics/native state of small protein structures.

Differential Segmentation

Uses a centrifuge to separate protein from lipids based on size. The complexes with the larger mass will be toward the top pf the tube. It is used for protein purification.

Protein Fractionation: Ion Exchange

TLC technique that has a charge on their beads. As the solution passes through a tube, the charged protein will attract or repel to the beads. Used for protein purification.

Protein Fractionation: Size Exclusion

Beads are like wiffle balls, where the larger stuff will pass faster while the smaller stuff will pass slower because stuck inside the beads. Protein Purification.

Protein Fractionation: Affinity

Beads will have a ligand on them that corresponds to desired protein. The protein would come out last because it’s attached to the ligand on the beads. Protein Purification.

SDS-Page

Gel for proteins, in which 1) Denature Protein, 2) Attach SDS once cooled 3) Run to red. This technique separates proteins based on size. (m/z ratio)

Western Blot

Uses antibiotics to identify known protein in solution. (1) Wash with Primary anitbody that attaches to protein of interest, (2) wash with secondary antibody that attaches to primary antibody. (3) wash with enzyme that will attach to seconday antibody and will have color as a substrate. Wanted protein will show up in color.

ELISA

Identifies and Quantifies known protein in solution. Wash w/Antibiotics in 96 well plate and look at color compared to a standard curve.

Isoelectric Focusing

Separates protein based on isoelectric point. Have a gel tube with different pHs, protein will stop moving once it’s pKa = the pH of the tube.

2D Electrophoresis

Has SDS page on one axis, Isoelectric focusing on another. Run a gel with both of them to separate protein from a bag of protein based on size and charge.

LC-MS-MS

Used to Identify sequences of unknown proteins. (1) run in a Mass Spec. with quadruples, in which desired m/z of protein will show resistance around the four poles.

(2) Throw m/z protein in a collision cell for nitrogen to chop it up into fragments

(3) throw fragments in another mass spec that will show resistance based on AA side chain

(4) can piece together AA sequence

NA Electrophoresis

Uses Gel as a sieve while electricity pulls DNA through based on m/z (size because charge is same for all nucleotides). DNA bands are identified/stained through pi-stacking of ETBr between nucleobases.

DNA Probing

Locates wanted DNA sequence within a chromosome via a “DNA Antibody” which is a outside complementary DNA strand that attaches to wanted to sequences and shows color.

Sanger Sequencing

Identifies Nucleotide sequences through ddbases that stop polymerization and gives color depending on the base. Have a ddbase at every nucleotide position and then align them and run a liquid gel to put sequence together.

Gel Shift Assay

Uses a native gel to determine NA-P interactions. Load samples and run to red. Samples that run slower are most likely NA-P bound.

Footprinting

Uses DNase to cut control and protein/DNA solutions. If NA-P interaction, then there would be missing bands on gel because protein would block DNase from cleaving in that particular location.

RT-PCR

Amplifies and Detects RNA transcripts by creating a cDNA strand, which then can be used for further testing. — Goes from single stranded RNA to one strand of RNA and one strand of DNA

Gene Chip

Compares transcription between two different cell types. In both cells: 1) Isolate transcripts 2) Perform RT-PCR to make them into cDNA 3) Add flourophore and denature them 4) Mix everything together and wash over chip with wells that contain reverse complements of certain proteins/metabolites. The colors of the wells indicate which cell type transcribes for that certain protein. — Qualitative Analysis!