Question

Can someone answer the question and provide a short explanation? Thanks!

Which of the following statements is NOT correct regarding protein seperation techniques?

A) Size-exclusion chromatography works on the same principle as polyacrylamide gel electrophoresis

B) Specific antibodies are required for immunoblotting, antibody affinity chromatography as well as immunoprecipitation

C) Two-dimensional gel electrophoresis is conceptually the same as combining gel filtration chromatography and ion exchange chromatography

D) Western blotting is a useful technique to isolate proteins for use in downstream assays

Answer 1

Polyacrylamide gel electrophoresis is a technique used to separate proteins. The proteins are separated on the basis of their mobility in a polyacrylamide matrix containing small pores under an electric field with the smaller molecules moving faster than larger ones. Addition of sodium dodecyl sulfate will conform uniform charge to the protein will remove any interference in mobility due to charged molecules present in the proteins.

Size exclusion chromatography is a chromatographic method which relies on separation of protein on the basis of size as well. Here the protein mixture is run through a column made up of porous beads composed of dextran polymers, agarose or acrylamide. Proteins with sizes smaller than the pore size of beads get trapped in the beads while larger ones get excluded out and move through the spaces between the individual beads and get eluted faster.

Affinity chromatography relies on the specific interaction between protein and the column to separate the protein. These interactions can be antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid. When a column containing an antibody specific to proteins is used it is known as antibody affinity chromatography.

Ion exchange chromatography separates proteins based on their affinity to an ion exchanger. This process is carried out at pH one unit away from the isoelectric point of the proteins as the at isoelectric point proteins will net charge of zero and hence will not bind to the ion exchanger.

Immunoblotting also known as western blots is used to identify a protein of interest following separation by PAGE. Proteins are transferred onto a nylon membrane from the gel and protein of interest is identified with help of antibody capable of binding to it. these antibodies or secondary antibodies capable of binding to protein-specific antibody are tagged with either chromogenic or fluorescent enzymes that in the presence of appropriate substrate can emit a signal thereby allowing for visualising the presence of protein. However, this method relies on fixing of separated onto a membrane and dousing them antibodies. This makes it unsuitable for use in downstream assays.

Immunoprecipitation also relies on using antibodies wherein protein antigen is precipitated out of the solution with the help of protein specific antibody bound to a solid substrate.

Isoelectric focusing is a method by which proteins are separated on the basis of the isoelectric point. Proteins are loaded onto an immobilised pH gradient. Upon applying electric fields proteins starts to move to either electrode based on their charge. However, when they reach pH corresponding to their isoelectric point their net charge becomes zero and gets immobilised.

2-D electrophoresis combine separation of proteins by isoelectric focusing with polyacrylamide gel electrophoresis. Here the proteins are first separated linearly on the basis of isoelectric point using a pH gradient. The molecules are then separated by applying an electric current in a perpendicular direction to initial separation.

Ans D) Western blotting is a useful technique to isolate proteins for use in downstream assays