Question

What the differences between IP, SDS-PAGE, Western and IF techniques (in vitro vs in situ experiments)?

Answer 1

IP ( Immuno precipitation) technique: The technique is based upon the specific antigen antibody interaction, In this technique a protein antigen is precipitated out by using an antibody specific for that particular protein antigen , the binding allows the precipitation to take place. By this technique isolation and concentration of a specific protein can be done from a sample containing several protein samples , therefore the technique is more of a SELECTION process.

SDS-PAGE :It is an electrophoresis technique which employs the use of an anionic detergent called SDS which denatures the protein structure. The technique is a variant of the Polyacrylamide gel electrophoresis (PAGE) , it forms cross linked matrix and pores .Separation of the PROTEINS BASED ON MOLECULAR WEIGHT can be achieved by this method.

WESTERN BLOTTING : Also known as protein immunoblotting , it is an ANALYTICAL TECHNIQUE FOR PROTEIN IDENTIFICATION .The proteins after separation by using Gel electrophoresis are transferred on to a matrix such as a nylon matrix , specific antibodies ( monoclonal and polyclonal antibodies) are used for the analysis process, the antibodies gets bound to the specific antigens or proteins.Suitable blocking agent is also used to avoid any non-specificic binding. Detection is done by use of X-ray film autoradiograph. The difference between Western blot and IP is that , Western blot targets protein which is denatured , while for IP the antibody used recognises the protein in its native (intact) state only.

IF Technique: The process or technique employs the separation of proteins based on the differences of their isoelectric point (pI) which is the pH at which an amino acid have 0 charge .It is also an electrophoresis technique , but in this case the proteins does't move in an electric field , rather movement takes plce under a pH gradient . The proteins gets separated out depending on their pI. Proteins having same molecular weight can be separated bu this method.