-properties of proteins are determined by conformation

-must prevent loss of conformation (denaturation) and chemical degradation

-must consider pH, contaminating enzymes (proteases) , temperature, reactivity of thiol groups

-method to quantify (assay) protein concentration

-method to direct protein of interest in a mixture of proteins (activity assays for enzyme or mobility in a el)

-tissue

-recombinant techniques

-sonication

-microfluidization

-Brands

.Sephadex

.Sephacryl

.Cellulose

.Biogel

-large molecules flow more rapidly though the column and emerge first because smaller volume is ccessible to them.

-a mixture of small proteins in a small volume is applied to column filled with pourous label because large protiens cannot enter intergral volume of beads and emerge sooner

[image]

[image]

Seperated based on net chare

-seect the appropriate resin based on protein chare at a crentain pH

above pI- net negative charge

below pI- net positive charge

1. Apply sample

2.Was with low salt concentration

3. Elute with an increasing salt gradient (0 to 1M for example) 

-powerful mean for isolating transcription factors

-flucose-binding protien attatches to glucose resues on beds

-Glucose-binding protins are released on additon of glucose

-ex: Many recombinant protiens are expressed with a 6 His tag added on t facilitate purification on a Ni2+ afinity column

-way of visulizing protiens

-protines not a the pI will move in n eletric field

-solid phases can be gels agrose or polyacdrylamide) or paper

-Electrophoretic mobilty epends on charge, size, pH, and voltage

-In SDS gel electrophoresis (denaturing gel eletrophoresis)

- a detergent sodium dodecylsulfate (SDS) and b-mercaptoethanol are added to the sample

-b-mercaptoehtanol

[image]

-samples move within a pH gradient in the gel (without SDS)until it reaches a position in the gell at which the pH is equalto the pI of the protein

-flat ben and open pourous gel ith gradient pH and protien will seperate via charge

is expressed as units

1 U= umoles/min

-comparge absolute purity by saying u/mg

-detrmine mass of sample

-make gradient of denisty in tube (layer)

-place in router in centrigue

-it would find its density

-fractionate based on density[image]

-hydrolyzing Amino Acid

-breaking small bonds

[image]

[image]

to label the N-ternminal residue

-one way to sequence

-protien in order

-used to tage in order to label

-once labelded can be hydrolysed

-the tg with label phenyl has a characterstic speperation can be identified

is a protien

-synthesized by an animal in response to the presence of a foreign substance called an antigen

Polyclonal antibodies

heterogenous mixture of anibodies, each specific for one of varios epitopes on an antigen

-ex. serum of rabit antiodies

Problem: antibody-producing cells isolated from an organism have shor life spans

Solution: derive cell lines form cancer multiple myeloma, which generates large number of clls of a single kind

Bigger solution:

-fuse shortlived antibody with immortal myeloma

-antigen injected in mouse

-spleen removed serveral weeks later

-pasma from spleen fused with immortal cells

-result

.produces identical ntibody specified by partent cell of spleen

Hybridoma cells are formed by the fusion of antibody-producing cells and myeloma cells

-the hybrid cels are allowed to proliferate by growing the in selective medium

-they are then screened to determine which ones produe antibody of desired specifity

. to detect presence of virus

Process

-antigen coated well

-wash

-specific antibody binds to antigen

-wash

-Ezme linked antibody binds to spcific antibody

-wash

-substrate is added and converted by ezyme into product

.detect antigen rather than antibody

-monoclonal antibody coated well

-wash

-antigen binds to anitbody

-wash

-second monoclonal antibody linked to enzyme binds to imobilized antigen

-wash

-substrate is added and cowashed by enzyme into produce

-permits directon of proteins seperated by gel electophoresis

-protiens on SDS-polyacrylamide gel are transferred to a polymer sheet (nitrocellulose) andstained with radioactive antibody

.a bond corresponding to the protein to which the antibody binds protein to which the antibody binds appear in the autoradio gram[image]

-a peptide hormone that stimulates water resoprtion

-a more stable syntheic aanolgo of this antidiruetic homone

. (matrix assisted base desorption-ionization)-(time of flight)

-how fast does molecule process