Western Blot Procedure (Updated 2011)
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In
the first stage of a Western
Blot,
PAGE (short for polyacrylamide
gel electrophoresis) is used to separate proteins. Polyacrilamide is
a polymer that forms a gel with pores that are small enough to
separate proteins. Two types of PAGE can be done. One is Native PAGE
which is performed under conditions of high pH, a condition that
causes almost all proteins to have a negative charge. Native PAGE
allows separation of proteins by their surface charge and their size.
The second type of PAGE gel is the type we will be using in lab. It
is called an SDS PAGE. In this system negatively charged SDS coats
individual proteins negating the charge on the proteins, the proteins
are also denatured by boiling them at 100 degrees Celsius in the
presence of a reducing agent. In this type of gel the proteins will
seperate on the gel based on size alone. A molecular weight can be
used to tell the sizes of various proteins.
In
the second stage of the Western Blot, Proteins from the gel are
transferred onto a membrane using electrophoresis. The proteins will
be transferred to the membrane in the same position as they are on
the gel. Finally, next week we will use antibodies to detect the
RUBISCO protein.
SETUP
Note: For Today's lab two groups will be working together. You will be sharing two 10 lane 10% acrylamide gels. On one gel you will load 30 ug of each of each of your protein samples to be stained using Comassie Blue. The other gel will be ran with 30 ug of each of your protein samples and then electroblotted. To keep you on track and enable you to try to get through this lab on time I made some suggestions below. If you are a group of 2 partner with a group of 3.
------PX (This must be done by 1 per table in the lab- have someone from a group of 3 take care of it). Prepare 1x Western Transfer Buffer for electroblotting and put the prepared buffer in the refridgerator. This buffer will be used later on during the Western Transfer but must be made and kept cold. Make this in the large flask at your work station. Please also make sure the flask gets washed out and put back at your work station at the end of the time today.
------ PX. Label three trays in hood for pure methanol , water and 1x Western transfer buffer (only required of first PX to get to this step)
------- P1 .Obtain tube of molecular weight marker from freezer and loading buffer from hood. Set up two tubes of molecular weight marker. You will need 10 ul of rainbow molecular weight marker and 15 ul of sample loading buffer. Your team will continue to need loading buffer.
------- P1 . Obtain approximately 500 ml of 1X running buffer (1x Western transfer buffer with 0.4% SDS) and have it available in a beaker. Obtain a razor blade, syringe with needle and 2- gels to get ready to set the gel-box up.
------ P2 and P3. Obtain your group's proteins from the -70 and allow them to begin to dethaw on ice. Label two 1.5 ml tubes for each of your samples. Poke holes with a needle in the top of the tube so that the lid will not pop open while your tube is boiling.
RUNNING THE GEL
------ P2 and P3. Aliquote out the appropiate amount of protein and QB buffer (you only have to use if you have low number of ul of protein) into the labeled 1.5 ml eppindorf tubes as was determined in the previous class. All tubes should contain the same number of ug of protein. You need two tubes for each protein sample. One sample (tube of protein) will be used to run the commassie blue stained gel. The second will be run on a gel to electroblot. To each tube add 15 ul of 3 x sample loading buffer (62.5mM Tris-HCl (pH6.8), 2% w/v SDS, 10% glycerol, 50 mM DTT and 0.1% w/v bromophenol blue.)
-------P2. Place your tubes in a boiling water bath for for 5 min.
____ (PX, P1, P3) Work with other team to create a loading diagram and start setting up the gel. Open gels and cut off bottom with razor blade. Remove comb and place in box with lane opening (small plate) to interior of box. Pour 1x running buffer into the center of the chamber and check for leaks to the bottom chamber as this will cause your gels not to run correctly. Once your apparatus has been confirmed as leak free you may fill the inner chamber up until it is just above the small plate and also fill the outer chamber about half way up. Remove the comb and use a syringe to wash out the wells several times with the 1X running buffer to remove any unpolymerized acrylamide from the wells.
------- (ALL) Take turns using the micropippete and loading your entire samples into the appropriate lanes.
------ (ALL) Assemble the top of the elctrophoresis apparatus and connect the wires to the power source.
------ (ALL) Run the blot at 200 volts for 45 min. or until the tracking dye in the sample buffer reaches the bottom of the gel. There will be a demonstration of the rest of the techniques used today at this time.
------ (ALL) Disconnect the power supply and disassemble gel apparatus.
------ (ALL) You will need to cut the gel so that you have some way to orientate it later. Perhaps by cutting the right top corner off.
MAKING THE GEL
In today's labortories, most scientist buy commercial gels rather than pour their own. We are going to pour one gel as a class so you have a chance to see how it is done but we will be using commercial gels to run our experiments. But to truely understand gel electrophoresis you should experience seeing a gel poured once so we are going to do it as a class while your gel is running (This time the entire class will participate- each student will assigned a number from 1-15).
Acrylamide Gels for protein electrophoresis are set up in two different layers the Running Gel layer which will seperate the proteins and the Stacking Gel layer which will allow the proteins to stack together immediatly after they enter the gel from the wells in which they were loaded. You will need to pour both of these layers individually before you can load your proteins onto the gel.
1. Set up the Gel Mold as Demonstrated in Class.
P1 and P2= Clean glass plates and help assemble apparatus
2. Prepare Running Gel as a class in a glass flask
P3= 8.15 ml water
P4= 3.75 ml running gel buffer (1.5 M Tris, pH=8.8)
P5= 2.8 ml 40% Acrylamid/Bis Solution 29:1
P6= 150 ul 10% SDS
3. P7= To this tube add 150 ul 10% Ammonium Persulfate, and P8= 6 ul of TEMED
4. P9= Mix with a glas pippete trying not to introduce air bubbles and then use the Glass Pippette to pour the gels to the correct level. Close the tube of unpolymerized solution and set asside.
5. P10= Carefully fill the rest of the gel chamber with Overlay buffer (Running Gel buffer with no acrylamide) to form an airtight space in which to allow the acrylamide to polymerize.
6. Allow the gel to polymerize for apporximately 1 hour or until the extra running gel mix in the tube you made is completely polymerized. For the purposes of this lab we will not go further but will talk about the rest of the steps in class and watch a video explanation of what is happening and why we have two layers to a gel (see: http://www.youtube.com/watch?v=snJMzL6KG_M.)
7. Remove the Overlay Buffer by blotting with a peice of filter paper.
8. Place comb in gel box and prepare stacking gel.
9. Again to prepare stacking gel, take appropriate tube from Stacking Gel buffer tube from fridge and add P15 = 25 ul 10% ammonium persulfate and 5 ul of TEMED.
P11= 1.3 ml stacking gel buffer
P12 = 3.2 ml water
P13= 0.5 ml acrylamide
P14 = 50 ul 10% SDS
10. P16 = Pour in the Stacking Gel Buffer with a pippette until entire chamber is filled. Allow to polymerize for approximately 1 hour.
-------P2. Transfer the gel to be stained to a plastic tray and submerge it in Comassie blue stain. Cover the box with saran wrap and shake it gently on the shaker overnight (wear gloves). Comassie blue will stick to all aromatic amino acids to help stain your protein.
-------(ONE GROUP MEMBER OR MORE). Tomorrow you will need to transfer this gel to destain (water) for 1 hour and then again to fresh destain (water). When gel is appropiatly destained (takes about 4 hours to overnight) you will want to come in and take a picture of the gel.
------ PX. Obtain western transfer buffer from fridge for your table (you made this at the beginning of class.
------ PX . Fill plastic Tray for 1X Western Transfer Buffer on table for each group.
------ PX. Fill three trays in hood for pure methanol , water and 1x Western transfer buffer (only required of first PX to get to this step)
------ P1. Obtain membrane and take to hood. Prepare the membrane once PX has filled solution boxes. Do not touch the membrane with your bare hands - use a forcips to handle it and make sure you have gloves on so proteins from your hands don't end up on the membrane.
------- P3- Get tray for 1X Western Tranfer Buffer and wait for Px to fill. When filled remove second gel and soak for 10 minutes in 1 X Western Transfer Buffer. We do this because gels put in methanol will shrink over time. You don't want the size of the gel to shrink during the blot, so soak it longer if the gel is continuing to shrink.
------ P3. Cut two pieces of Whatman filter paper also the same size as gel. Also go and get a glass pastuer pippette.
------ (ALL) After all of the above are prepared you are ready to prepare the Gel Sandwich:
Which direction should the gel run to transfer the protein from the gel to the membrane?
------ (All) Put a magnetic stir bar inside tank and run on magnetic plate in the cold room.
------ (All) Gels will be ran in the cold room next door overnight at Constant current (250 mA)
------- (Decide who is responsible for coming back tomorrow morning and taking the membrane off and performing the following steps.
When you remove the membrane, use a pencil to label the location of the wells and the initials of the group members on the side that the protein was transferred to or put some sort of label on it so that you know the orientation of the gel and where the lanes are and whoose gel it is. Do not let the membrane dry out!!
updated March, 2012- TKR