Chem lab final

Buffer

A solution that resists changes in pH upon the addition of small amounts of acid or base.

ka

ka=(H+)(A-)/(HA)

pKa

The negative logarithm of the acid dissociation constant (Ka), indicating the strength of an acid.

Henderson-Hasselbalch equation

An equation used to calculate the pH of a buffer solution, given by pH = pKa + log([A-]/[HA]).

Beer lambert equation

A=€cl

stronger acid = ___ pka

smaller

Lysozyme

An enzyme that catalyzes the hydrolysis of glycosidic bonds in peptidoglycan, leading to cell wall rupture.

Peptidoglycan

A polymer consisting of sugars and amino acids that makes up the cell wall in bacteria.

lysozyme

cataylzes the hydroylsis of Beta 1 to 4 glycosidic bonds between NAG and NAM

Ion-Exchange Chromatography

A technique used to separate molecules based on their net charge.

Homodimer

A protein composed of two identical subunits.

Cation exchange

A method using negatively charged resin to bind positively charged proteins. (CM sepharose) -positive charged will bind

Anion exchange

A method using positively charged resin to bind negatively charged proteins. (DEAE sepharose)- negative charge will bind

pH < pI

protein has positive charge

pH > pI

protein has negative charge

lysozyme PI

very high -10.7

Biuret method

A colorimetric assay for detecting the presence of proteins based on the formation of a colored complex with copper II ions.
Color changes from light blue to dark purple

not very sensitive

ELISA

A technique that uses antibodies to detect the presence of specific antigens in a sample.

function of BSA in ELIZA

excess inert protein that locks sites not covered by protein

Michaelis-Menten equation and plot

lineweaver burke plot

Salt precipitation

A method used to purify proteins by adding salt to precipitate proteins out of solution. disrupt hydration sphere, salt pulls water molecules from surface of protein, forcing protein molecule to aggregate

-maintains protein native structure

how is got activity indirectly measured

• oxaloacetate reacts with NADH to create L-malate and

• NAD+, NADH absorbs at 340 but NAD+ does not

Heat denaturation

The process by which proteins lose their structural integrity and function due to high temperature. irreversible

IU/Ml equation:
Specific Activity equation:

Total GOT activity equation:

%yield fraction:

Degree of purity:

Coomassie Blue

A staining dye used in electrophoresis. we use with acid/methanol mixture to fix proteins to the gel to prevent protein form diffusing in gel or being washed out

BME (Beta-Mercaptoethanol)

A reducing agent used to break disulfide bonds in proteins during SDS-PAGE.

SDS-PAGE

A technique that separates proteins based on their size by using polyacrylamide gel electrophoresis with sodium dodecyl sulfate.

Native PAGE

Polyacrylamide gel electrophoresis that maintains the native structure and charge density of proteins.

Polyacrylamide gel

A cross-linked polymer of acrylamide and bis-acrylamide used as a medium for gel electrophoresis to separate proteins based on size

higher % acrylamide to seperate ___ (big/small) proteins

small- dense matric- restrict movement of the large proteins

SDS

used to break non-covalent interactions (all become neg charged and linear)

BME

• reducing agent to reduce (break) disulfide bonds

reducing conditions

• BME+SDS

• disulfide bonds broken

• non-cov broken

non-reducing conditions

• SDS only

• disulfide not broken

• non- cov broken

bromophenol blue in sds-page

doesn’t stain proteins, just shows progress of electrophoresis