Background Western Blot Protocol Western blotting is an analytical technique that involves transferring the proteins that have separated through SDS PAGE to a nitrocellulose membrane Good 53 Western blotting uses antibody antigen interactions in order to identify an unknown protein In order to transfer the proteins from the gel that was made through SDS PAGE to the nitrocellulose membrane the gel is placed right next to the Nitrocellulose in a blotting sandwich A blotting sandwich consists of gel nitrocellulose Scotch Brite pads and filter papers The materials are layered on top of each other and locked together The blotting sandwich is immersed in buffer and an electric current is ran through it The negatively charged proteins are drawn towards the Nitrocellulose which is on the positive pole of the blotting sandwich Proteins are transferred from the gel to the nitrocellulose because Nitrocellulose can be manipulated in ways that the gel cannot Good 53 The advantages of transferring the proteins to the nitrocellulose include the proteins becoming more stable being able to be stored for a long period of time and proteins that are on the nitrocellulose are more accessible to primary and secondary antibodies The exact mechanism for protein binding to the nitrocellulose is unknown but it is theorized that the binding is due to hydrophobic interactions which are facilitated by the methanol in the buffer that the gel and nitrocellulose are immersed in Good 54 Nitrocellulose membranes are covered with active binding sites which makes it difficult to identify a specific protein To find the specific location and identity of a protein Nitrocellulose must first be blocked by non specific proteins such as the ones found in milk To determine the identity of the protein nitrocellulose is exposed to an antibody The first antibody that the proteins on the nitrocellulose bind to is called the primary antibody Then a secondary antibody is added to bind to the primary antibody The secondary antibody is important because the enzymes on the secondary antibody can be used to yield the location of a specific protein on the nitrocellulose The purpose of using western blotting in this experiment is to determine our protein s molecular weight and composition and find the identity of the protein through binding of the nitrocellulose membrane to specific antibodies In this experiment nitrocellulose will bind to one of the primary antibodies that we picked which will help us determine the identity of our unknown protein Protocol SDS PAGE 1 Obtain 50 L of your unknown protein sample 2 Obtain an Eppendorf tube and label it as protein sample 3 In the tube add the accurate amount of unknown Protein 20 L with the use of a micropipette The amount you should add to the tube can be viewed in the Dilution chart below Be sure to change the micropipette tip when you are adding a sample to the tube to avoid contamination 4 Next micropipette the accurate amount of PBS 20 L to the tube which can also be 5 Add 40 L of sample buffer to the tube and mix the solutions in the tubes by flash viewed in the Dilution chart spinning them in a centrifuge 6 Place the tube into a thermocycler and boil the samples at 90 95 for 5 minutes Then 7 To prepare the gel remove it from the package and remove the tape lined at the bottom of flash spin the tube in a centrifuge the gel 8 Carefully take out the comb that is lodged into the gel by using even pressure on both sides of the comb to not break the gel 9 Load the gel into the gel electrophoresis apparatus and fill the center of the apparatus with 1X electrode buffer and the sides up to the line that says 2 Gels 10 Carefully load 10 L of the DNA standard into the first well Then Add 15 L of sample buffer into the second well Lastly add 15 L of the protein dilution into the third well Follow this order for 2 more protein dilutions and in the tenth well add 15 L of sample buffer 11 Add the top of the apparatus making sure to align the red and black sides and plug the apparatus into the power bank 12 Run the gel for 5 minutes at 100 volts then run it at 200 volts for 30 minutes Blotting the Gel 1 Place the black side of the transfer cassette at the bottom of the transfer buffer solution 2 Next layer on one Scotch Brite pad then layer the scotch brite with a filter paper sheet 3 Place the gel on filter paper and cover the gel with a sheet of nitrocellulose 4 Layer a piece of filter paper on top then layer a Scotch Brite pad on top of the filter paper 5 Before you close the red side of the transfer cassette gently roll a pipet over the top of the sandwich to remove any bubbles trapped between any of the layers Close the red side of the transfer cassette over the sandwich and lock it in place 6 Place the transfer cassette lock side at the top into the blotting chamber The gel should be on the black cathode side while the nitrocellulose is at the red anode side 7 Fill the apparatus up to the 4 gel line with cold transfer buffer Place an ice pack and stir bar into the apparatus as well 8 Place the gel apparatus on a stir bar machine Run the gel for 45 minutes at 100 volts to transfer the gel proteins to the nitrocellulose paper 9 Disassemble the protein sandwich and place the nitrocellulose paper into a Ziploc bag Incubate the nitrocellulose for at least 24 hours at 37 Antibody Detection 1 After incubating the nitrocellulose membrane place the membrane into a weigh boat Block the membrane by pouring 5 milk in TBST until the membrane is completely submerged 2 Rock the weigh boat on a rocker for 10 minutes Then carefully discard the liquid in the weigh boat into the sink 3 Next wash the nitrocellulose membrane 3 times with 1 milk in TBST a Pour 1 milk over the nitrocellulose membrane until it is completely submerged b Rock the weigh boat for 5 minutes on the rocker c Discard the liquid into the liquid waste d Repeat steps a c two more times for a total of three times 4 After washing cut the nitrocellulose paper into 3 portions to test the antibodies that have been chosen 5 Place each portion into its own weigh boat and add 10 mL of 1 milk in each of the weigh boats Add 10 L of the different primary antibodies to each portion and label which portion contains which antibody 6 Rock each weigh boat for 45 minutes 7 Repeat step 3 8 Add 10 mL of 1 milk to each weigh boat Then add 10 L of the different secondary antibodies to each portion and make sure to properly label them Rock the portions for 30