Methods and Techniques Discussion => Western Blotting (WB) => Topic started by: Jan on November 29, 2008, 02:47:18 AM

Title: I need some aplications for of Western blots for my assignment pls help
Post by: Jan on November 29, 2008, 02:47:18 AM
i am doing an assignment on western blot i am a 1st year B Sc bio/chem university student.
I have found may info on the technique per se but when look up on journals[nature, science] the practical use of western blotting is extremely complex and i do not understand how it is used.
Can anyone please direct me to any articles/papers of scientific lit. about a relatively simple application of western blotting. or any other help because i need to include recent discoveries/ applications in my assignment.
thanks for your time
Jan
Title: Re: I need some aplications for of Western blots for my assignment pls help
Post by: Cardio on December 01, 2008, 03:14:06 PM
Quick suggestion,

Look up 2-D gels which is a way to identify proteins by molecular weight and charge. Most westerns are 1-D but this will give your report more depth.


Title: Re: I need some aplications for of Western blots for my assignment pls help
Post by: MT Scientist on December 03, 2008, 05:57:38 PM
Western blotting in may cases is a combination of techniques.  Process samples (proteins), run samples on a PAGE gel (SDS or Native) and transfer to a membrane (PVDF or Nitrocellulose).  This transfers the proteins separated by size/charge or both ot the membrane.  The unbound portions (i.e. any part of the membrane that doesn't have protein bound to it) of the membrane are blocked with any number of substances (skim milk/BSA/Casein/etc) and the blot probed with an antibody of interest.  The bound antibody is labeled, usually by using a second antibody that labels the FC portion of the first antibody (and depends also on the species from which the first antibody is derived) that is tagged with some sort of molecule that will react with your substrate of choice to show the signal.

EX.  Antigen of interest detected by an antibody made in mouse to the antigen, the bound mouse antibody is labeled by a goat anti-mouse antibody labeled with HRP (horseradish peroxidase).  The substrate is added and the HRP cleaves the substrate to give a signal (visible/chemiluminescent) that is detected by an appropriate instrument.

"Western" refers to the detection of a protein with an antibody, as opposed to "Northern" or "Southern" blotting which are used for RNA and DNA detection respectively.

I would suggest searching on line for a manual on PAGE and western blotting rather than in a specific journal.  It will explain the techniques in a bit more detail.....