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Geneomic Library Construction Using Plasmids
Spin Trapping Free Radicals
Chrome azurol S (CAS) assay
Denaturing Gradient Gel Electrophoresis
Most probable number
Fatty Acid Methyl Ester Analysis
Acid Guanidinium-Thiocyanate-Phenol-Chloroform Extraction of Total RNA
Phospholipid-Linked Fatty Acid (PLFA) Profiling
Polymerase Chain Reaction
Testing Soil Salinity-conductivity meter
Chlorofom Fumigation Direct Extraction
Barbara B Fowler
There are several different bloting techniques that can be used to detect different types of macromolecules. One of which being the Southern blot, which is used to detect DNA then there is the Northern blot which is used to detect mRNA. The focus of this is going to be on the Western blot which is used to detect protein. ( genetic). Proteins are detected based upon their ability to bind to specific antibodies.
Western blot is also commonly called an immunoblot which originated in the laboratory of Gerorge Stark at Standford and given the name of Western blot by W Neal Burnette. THe western blot is often also requires running an SDS PAGE gel ( sodium dodecyl sulfate polyacramid gel electrophoresis) which is a technique used to seprate proteins according to their electrophoretic mobility ( a function of length of polypeptide chain or molecular weight)
In order for this to work samples are mixed with SDS which is an anionic detergent which denatures secondary and no disulfid linked tertiary structures and applies a negative chare to each of the proteins. The SDS has a specific binding ration of 1.4g per 1.0g protein, this allows for the proteins to run through the gel and sort depending on size.
methods and materials
first of all an SDS PAGE gel needs to be made ( or bought but making your own is cheaper and easy) . There is an apparatus available from BioRad as well as a Protocol. Depending on the protein of intrest will depend on the precent acrylimide needed, the most common is 12-15% acrylamide.
next samples for the western blot need to be prepared to a concentration of 0.1micro gram to 10micro gram of water ( not tap water, it contains to many contaminates MilliQ water or DI water) . The samples are then loaded into the gel along with a molecular weight marker. the apparatus is then filled with buffer and hooked up to a power source and ran at a constant voltage of 200v for 45 minutes.
During that time a polyvinylidone flourid membrane (PVDF membrane) is prepared by carefully transfering it to a plastic box with a small amount of methonal in it and then the membrane is rinsed with the methonal and drained, then transfer buffer fills the box with the membrane in it and it is placed in a fridge, until it was time for it to be used.
once the gel was done runing the apparatus was disassembled , after that the "sandwhich" needs to be assembled
in this order in the western blot hinge cover ( making sure it is done in PVDF transfered buffer) sponge pad, whattman paper, the gel making sure it is faced down, the PVDF membrane, 2 piece of whattman paper , the sponge pad. The hinge cover was then closed and placed into the apparatus with a stir bar at the bottom.
From there the apparatus is moved to a cold room of about 4 degrees C this is done because the apparatus tends to heats up and this helps it to stay cool. my tip is to place the apparatus into a plastic box so it wont leak all over. The apparatus is then hooked up to a power source and ran at a constant voltage of 100v for 2 hours.
when the two hours are up the sandwhich is disassembled , the gel should be clear and the molecular weight marker should be on the PVDF membrane , discard everything else and allow te PVDF membrane to dry.
once the membrane is dry then antibodies can be used to detect protein present.
the anitbody is mixed with 5% milk in western blot buffer and poured over the membrane and left to incubate for 1 hour. The antibody is mixed with milk so the milk will bind to all other proteins preventing the antibody from binding to the wrong protein.
then a second antibody can be applied in the same manner but only for 30 Minutes
then the membrane is washed 3 times with Western blot buffer and then developed by using a developer like ECL Plus following manufactuers directions. From there then the membrane can be photographed using a Versa doc imager or by developing in a dark room , but that is a different method of developing ( alot more complicated and time consuming)
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