protein analysis techniques + downstream applications

proteins

restricted with isolation procedures; unstable

low

quantity of the protein of interest tends to be quite _ when it is isolated

FALSE

T or F: proteins can be amplified like nucleic acids

Lysing the cells & tissues

first phases in cell fractionation; its goal is to disaggregate the cells and break them open with minimal damage to the cellular fraction of interest

heat treatment, homogenization, sonication

physical methods

treatment with detergent solution

chemical methods

disrupt the tissues and cells

first step in the physical disruption

homogenization

used to rupture plasma membrane of cells to release its contents

homogenate

product of homogenization

high frequency sound

(homogenization) used to break cells

mild detergent

(homogenization) used to make holes in the plasma membrane

high pressure

(homogenization) used to force cells through a small hole

shear cells

(homogenization) _ between a close fitting rotating plunger and the thick walls of a glass vessel

mechanical disruption

use of rotating blades to grind and disperse substantia

specimen; polytron

polytron

draws the tissues into a long shaft containing rotating blades and can be used with samples as small as 1 mL

Liquid homogenization

most common technique under the lysis technique under physical disruption; dounce, potter-elvehjem, and french press

dounce homogenizer

consist of a round glass pestle, manually driven into a glass tubes

Potter-elvehjem homogenizer

manually

mechanically driven pestle that is shaped to fit a rounded

conical vessel

french press

used to extract

isolate the protein; consists of a piston; expensive and is the method of choice for breaking bacterial cell mechanically

piston

(french press) used to apply high pressure to a sample volume of 40-250mL

2

(french press) how many passes are required for efficient lysis

freeze-thaw

commonly used to lyse mammalian and microbial cells, protozoan cysts, oocysts, and fungal cell wells; used to obtain recombinant proteins

mortar and pestle

most basic and most common; tissues are subjected in liquid nitrogen then mortar and pestle

plant proteins and DNA

mortar and pestle is used for? fastest and most efficient also

sonication

pulsed, high-frequency sound waves; apparatus with a vibrating probe, immersed in a liquid suspension

sample obtained in sonication

microscopic vapor bubbles

(sonication) mechanical energy is released from the probe and initiates the formation of _ that causes shock waves to radiate to the sample

generates heat and damages the protein of interest

disadvantage of sonication

applying in multiple short bursts

remedy (sonication)

western blot

utilizes Abs to determine individual proteins within a cell or tissue lysate

ELISA

target Ag or Ab is captured in samples using a specific Ag or Ab; enzyme reaction with its substrate

light absorption

measured and then converted to numerical values

ELISA plate reader

converts to numerical values