Chapter 8 - SDS Page (copy)

detergent, secondary, tertiary, quaternary, hydrogen, hydrophobic, ionic, uniformly, folded, linear, negative, negligible, negative, covalently, negative, negatively, linear

Sodium Dodecyl Sulfate (SDS):

• a _____ that helps eliminate of the _____, _____, and _____ structures

• disturbs _____ bonds, _____ interactions, and _____ bonds

• binds _____ to protein

• converts _____ protein to a _____ molecule with a net _____ charge

• intrinsic charges of protein become _____ compared to _____ charges contributed by SDS

• denatures (at least partially) proteins and peptides

• binds non-_____

  • gives the proteins/peptides an overall _____ charge as SDS is _____ charged

• charged _____ molecule → can be separated in an electrical field

SDS, denatures, hydrogen

Why do we heat the sample?

• opens up the protein to allow the _____ to gain better access

  • heat _____ (breaking the _____ bonds)

Beta-mercaptoethanol (BME)

breaks disulfide bonds at equilibrium

Dithiothreitol (DTT)

permanently breaks disulfide bond

disulfide, equilibrium, disulfide, permanently, disulfide

Beta-mercaptoethanol (BME):

• breaks _____ bonds

• volatile (evaporates)

• reaction forms an _____

  • _____ bonds are continually breaking and reforming

Dithiothreitol (DTT):

• not as volatile as BME

• DTT is altered during reaction with disulfide

  • Sulfhydryl groups stay reduced

  • DTT _____ breaks _____ bonds

glycerol

increases density (sample sinks to bottom of well)

bromophenol blue

• to visualize the sample

• dye front runs ahead of proteins (monitor progress)

  • acid - base indicator (blue at 4.6+; if yellow = BAD)

acrylamide, agarose

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE):

• electrophoresis in _____ gels (not in _____ gels)

• used to separate and identify proteins from a protein mixture such as a cell lysate

acrylamide, N,N’-methylene bisacrylamide (Bis), ammonium persulfate (APS), TEMED (catalyst)

the polymerization reaction of acrylamide:

• polyacrylamide gels are formed by chemical polymerization of _____ and the cross-linking reagent _____ _____ (_____)

• polymerization is initiated by _____ _____ (_____) and _____ (______)

inert, stable, transparent, small, smaller

advantages of using polyacrylamide gels:

• chemically _____

• _____

• _____

• _____ pore size = suitable for _____ molecules (e.g. proteins)

2

polyacrylamide gels have _____ layers

stacking, resolving

polyacrylamide gels have 2 layers:

• _____ gel

• _____ gel

stacking gel

deposits all proteins in a tight band so that they can start migrating through the resolving gel simulataneously

resolving gel

separates proteins based on their molecular weight

6.8, 4-5, 8.8

stacking gel:

• pH _____

• _____-_____% acrylamide

resolving gel:

• pH _____

• can vary % of acrylamide depending on size of target protein

acrylamide, crosslinker (Bis)

• investigators can control the pore size of the resolving gel by adjusting the concentration of _____ and _____ (_____)

inversely, higher, lower

pore size is _____ proportional to acrylamide and Bis crosslinker concentration

• smaller proteins require a _____ acrylamide % in resolving gel (smaller pores)

• larger proteins require a _____ acrylamide % in resolving gel (larger pores)

molecular weight, lower

• % acrylamide gel depends on the _____ _____ of the protein of interest

• optimum separation occurs in the _____ half of the gel

resolving, simultaneously, glycine, zwitterion, slowly, faster, negative, voltage gradient, negatively, quickly,

stacking gel:

• deposits all proteins in a tight band so that they can start migrating through the _____ gel _____

• _____ (running buffer component) is mostly in its _____ state (no net charge) at pH 6.8 and moves _____ through the stacking gel

• Cl- ions (from Tris-HCl) move _____ because of their _____ charge

• the leading Cl- ions and the slower moving glycine zwitterions create a _____ _____

• the electromobility of proteins is somewhere in between Cl- and glycine zwitterions

• proteins are concentrated and stacked into zone between Cl- and glycine zwitterion front

• glycine ions become mostly _____ charged as they enter the resolving gel (pH 8.8) and _____ pass the protein layer, depositing the proteins in a tight band at the top of the resolving gel

molecular weight

resolving gel:

• once the proteins enter the resolving gel, they will be separated based on their _____ _____

relative mobility/retention factor (Rf)

• movement of a type of polypeptide through a gel relative to other protein bands in the gel

• distance migrated by a band divided by the distance migrated by the dye front

log MW, migration distance (Rf)

graph of _____ _____ of the standards in protein ladder vs relative _____ _____ (_____) can be utilized to more precisely determine the molecular weight (MW) of unknown proteins using the graph equation

Coomassie Brilliant Blue, Silver Staining, Fluorescent Stains

3 ways to stain gel:

• _____ _____ _____

• _____ _____

• _____ _____

widely, low, quantitative

Coomassie Brilliant Blue staining:

• most _____ used

• _____ sensitivity

• _____

• does not modify target proteins (compatible with mass spectrometry)

silver, functional, great, quantitative, expensive

Silver staining:

• _____ ions bind strongly with certain protein _____ groups

• _____ sensitivity

• not _____, since staining intensity does not bear a linear relationship to protein mass

• _____

most, most

Fluorescent Stains:

• fluorescent dye binds to protein and is activated by specific wavelength to emit fluorescent light

• _____ sensitive

• compatible with mass spectrometry

• _____ expensive

• requires special equipment to view

Western blot analysis, Mass spectrometry

experimental procedures that are commonly performed following SDS-PAGE:

• _____ _____ _____

• _____ _____