SDS - PAGE

SDS - PAGE : Sodium Dodecyl Sulfate - PolyAcrylamide Gel Electrophoresis

purpose: to separate proteins of different molecular sizes for analysis

DNA molecule unit for measuring size - base pair (bp)

Protein molecule unit for measuring size - Dalton (Da)

• one dalton is defined as the mass of a proton - 1.66×10^-24 g

• most proteins have masses on the order of thousands of daltons - kilodalton (kDa)

• avg mass of amino acid = 110 daltons

• molecular weight of protein = number of amino acids x 110daltons

materials

• Acrylamide/Bis-acrylamide

  • Acrylamide (a white crystalline powder) is a neurotoxin - while dissolving in water, acrylamide monomers slowly auto-polymerise into long straight chain of polymers

  • Bis-acrylamide crosslinks acrylamide polymer chain into a network

  • pore size of gel is determined by the total amount of monomer (acrylamide) present and the amount of cross linker (bis-acrylamide) - generally lower percentage of acrylamide will be used to separate larger proteins

• Sodium dodecyl sulfate (SDS)

• TEMED and Ammonium persulfate (APS)

  • TEMED used as catalyst to speed up the gel polymerisation reaction

  • APS provides free radical to initiate polymerisation

• Gel casting tray and comb

• electrophoresis chamber

• coomassie blue

protein prep

different protein molecules in their native state have different shapes and charges

hence the first step of SDS-PAGE is to boil the proteins in SDS and loading dye.

boiling denatures the proteins into linear form

• boiling disrupts weak bonds/interactions between amino acids e.g. hydrogen bonds, hydrophobic interactions

SDS denatures proteins and confers -ve charge

• SDS molecules carry -ve charge and binds strongly to the amino acid

• polypeptide chain unfolds as the neg charges btwn the amino acids are alike and repel

• proteins lose their 3D conformation

proteins need to be denatured into linear form as in their native states, proteins have different 3D shapes/conformations, their =shapes will affect the speed of proteins travel in SDS PAGE hence for speed of protein migration proportional to solely molecular weight it is necessary to denature proteins into linear form

proteins need to be given negative charges as different proteins or amino acids carry different charges in their native state. SDS is required to confer the negative charges to proteins so that all proteins will move towards the anode/positive pole during SDS PAGE

electrophoresis

• boiled protein samples are loaded into gel. Glycerol helps to “sink” the sample into the wells

• Gel submerged into running buffer

• proteins of different sizes squeeze through the pores in the gel matrix (low mw moves further towards the anode side)

analysing results

• Gel is soaked in Coomassie blue solution which binds strongly to all proteins

• unbound dye is removed by extensive washing of th gel

• blue protein bands can be located and quantified since the amount of bound dye is proportional to the protein content

• stained gels can be dried and preserved/photographed

results can find

• how many types of proteins are there in the sample

• mw of proteins

• how pure is the protein of interest

• how much protein is there

• differences in the proteins from different sources

tutorial ans

similarities btwn agarose gel electrophoresis and SDS PAGE

• both separate molecules based on molecular size - smaller fragments move faster through the pores of the gel matrix and therefore further than larger fragments

• both use the gel matrix as the molecular sieve to separate molecules

• in both systems, molecules move from negative terminal to the positive terminal when subjected to an electric field

• staining is required for visualisation of bands

• both require use of loading dye for samples to be loaded into the wells

• both require samples to be linearised before gel electrophoresis