BS2520 - Protein Structure and Function Lecture 1 Introduction to SDS-PAGE and Western Blotting SDS-PAGE Gel electrophoresis is a method of separating charged molecules through a gel. Larger proteins are trapped by the gel matrix whereas smaller proteins can more easily migrate through the pores. Acrylamide gels (used in SDS-PAGE) form pores the size of many proteins which allows molecular sieving to occur. - There are cross links with bisacrylamide. Native gels - separation will depend on a combination of size and charge. SDS-PAGE gels are more commonly used than native gels. Proteins are coated with sodium dodecyl sulphate, SDS. SDS is a negatively charged detergent which denatures and linearises proteins, coating them in a negative charge. Folding of secondary and tertiary proteins is removed (linearising). SDS-PAGE separation occurs by size only as all proteins are negatively charged, with an almost equal charge/mass ratio. B-mercaptoethanol is added to protein samples before they are added to the gels. It works to remove disulphide bonds, allowing the protein to become linear. B-mercaptoethanol is added in the loading buffer. Together, B-mercaptoethanol and SDS-PAGE prepare proteins. S 20 Mercaptoethanol denatures protein SDS SDS binds protein stoichiometrically 1 Smell molecule moves more quickly through gel Large molecule moves more slowly through gel
Laemmli electrophoresis (discontinuous gel system). There is a stacking gel and resolving gel. Cathode - Stacking gel Running gel Anode + U - Running gel is the resolving gel. Sample wells Buffer The stacking gel helps proteins to all enter the resolving gel at the same time. Plastic frame This works by having two different compositions in the two gels. The stacking gel is about ff% acrylamide and the resolving gel is 8-12% acrylamide. Buffer The buffer is Tris-glycine with a higher pH of 8.3. - This change in pH is key - there is a shift in pH which changes glycine from the zwitterion state to the negatively charged state. The glycine in the buffer is important for protein stacking. It can exist in 3 different states, a cation, zwitterion, or anion. The proteins on the gel are stained with Coomassie Blue. The stain binds to proteins through ionic interaction. It is a non-specific stainft it stains all proteins present. This means you are unable to identify any specific protein. Western Blotting Electroblotting is the transfer of proteins from the gel to a membrane. The membranes used are nitrocellulose of PVDF, both bind proteins very well. The membrane can then be used for biding with an antibody to identify specific proteins. The gel is placed adjacent to the membrane. A voltage gradient is applied and negatively charged proteins migrate towards the anode from the gel to the membrane. This process takes place in a blotting tank. + Blotting paper soaked in buffer Gel Membrane 1 Blotting paper soaked in buffer
Immunodetection Membranes are incubated with antibodies to detect specific proteins. Antigen binding-site Antigen binding-site NH ·Light chain Disulfide bonds Hinge region Heavy chain COOH The antibodies come from cell cultures or animal