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1、WESTERN BLOT,Hongbo-chen 2011/10/31,1 BACKGROUND,This Technique was discovered by Dr. Douglas Lake of the University of Arizona School of Medicines Department of Microbiology and Immunology. Dr. Lake is now studying how the immune system can be harnessed to attack cancer.,2 What is Western Bolt?,A t
2、echnique in which proteins are separated by gel electrophoresis and transferred to a membrane sheet. A specific protein is then identified through its reaction with a labeled antibody. So called since it has some similarity to a Southern blot.,Difference Among Southern Northern Western Blot,Southern
3、 Blot is for DNA detection. Nouthern Blot is for RNA detection. Western Blot is for Protein detection.,3. Why we have to use it?,We can use this technique to identify a target protein in a complex mixture, and we can also use it to measure its expression level.,Several commonly used technique to com
4、pare with the Western blot,The basic principles of Western Blot,Under the role of the electric peptides separated by gel electrophoresis(usually SDS-PAGE) transfer from the gel to one kind of solid support body, and then use this peptide-specific antibodies to detect.,SDS-PAGE, sodium dodecyl sulfat
5、e polyacrylamide gel electrophoresis（十二烷基磺酸钠-聚丙烯酰胺凝胶电泳）, is a technique widely used in biochemistry, genetics and molecular biology: to separate proteins according to their electrophoretic mobility(电泳迁移率),a function of length of polypeptide chain or molecular weight. to separate proteins according t
6、o their size, and no other physical feature.,SDS (sodium dodecyl sulfate) is a detergent (soap) that can dissolve hydrophobic（疏水性） molecules but also has a negative charge (sulfATE 硫酸盐) attached to it.,Before SDS: Protein (pink line) incubated with the denaturing detergent SDS showing negative and p
7、ositive charges due to the charged R-groups in the protein. After SDS: SDS disrupt hydrophobic areas (Hs) and coat proteins with many negative charges which overwhelms any positive charges the protein had due to positively charged R-groups. The resulting protein has been denatured by SDS (reduced to
8、 its primary structure-aminoacid sequence) and as a result has been linearized.,PAGE,If the proteins are denatured and put into an electric field (only), they will all move towards the positive pole at the same rate, with no separation by size. However, if the proteins are put into an environment th
9、at will allow different sized proteins to move at different rates. The environment is polyacrylamide. the entire process is called polyacrylamide gel electrophoresis (PAGE).,PAGE,Small molecules move through the polyacrylamide forest faster than big molecules. Big molecules stays near the well.,The
10、end result of SDS- PAGE has two important features: 1) all proteins contain only primary structure & 2) all proteins have a large negative charge which means they will all migrate towards the positive pole when placed in an electric field.,4. How to do it?,Separate proteins by gel electrophoresis. T
11、ransfer proteins onto a membrane Wet tank transfer system Semi-dry transfer system Identify proteins Immunodetection,Equipment,Rule of thumb:The smaller the size of the protein of interest, the higher the percentage. The bigger the size of the protein of interest, the lower the percentage.,(1). Samp
12、le preparation,Protein denaturing SDS , mercaptoethanol（巯基乙醇） and heat (boiling 5min) breaks disulphide bonds（二硫键） - disrupts secondary and tertiary structure. Linear protein will then run at the correct molecular weight. May also make antibody epitope more accessible.,(2). electrophoresis,Load 20 L
13、 sample into the appropriate wells using a micropippete. Run the gel at 100 volts for 4-5 h ,or until the tracking dye in the sample buffer reaches the bottom of the gel.,(3). Running the gel,A negative charge is applied to the buffer in the inner chamber, to which the top of the gel is exposed,A po
14、sitive charge is applied to the buffer in the outer chamber, to which the bottom of the gel is exposed,Proteins (which are negatively charged) travel down toward the bottom (positive) electrode,The progress can be monitored by watching the dye travel toward the bottom of the gel,Smaller proteins mov
15、e more quickly through the gel. Proteins separate out According to size,Proteins negatively charged, move towards the anode.,Samples are pipetted into the wells,Stain,Coomassie Brilliant Blue,PonceauS red,(4). Transfer to the membrane,The gel is placed against a special membrane that binds proteins,
16、Choice of membrane: PVDF (Polyvinylidene fluoride) membrane often gives less background than nitrocellulose（硝酸纤维素膜） Larger molecular weight proteins both types of filters works well. If less than 10 kDa nitrocellulose should be used - much higher binding capacity.,(5). Blocking,Fat-dry Milk or BSA 5% for 1 to 2 hours. The membrane is incubated with a protein block (usually either BSA or milk) to occupy the spaces on the membrane that are not yet occupied by protein. This preven
