![]() (DAB is most sensitive for immunocoupled horseradish peroxidase. Developing: (Use of chromomeric substrate- 3,3’-diaminobenzidine, DAB) – Wash in PBS with 0.1%Tween20, 3 X, and 10 min of each time 5. Incubate with secondary antibody (peroxidase-conjugated goat anti-mouse IgG, etc.) following the manufacturer’s instruction, usually 1:200 to 1:2000 1 hour at room temperature Wash in PBS with 0.1%Tween20, 3 X, and 10 min of each time (Optimal antibody concentration must be determined experimentally 1:500dilution is good starting point.) Incubate with Primary Antibody in 1%BSA in PBS for 1 hour at room temperature or overnight at 4C by shaking. Wash in PBS with 0.1% Tween20, 3 X, and 10 min for each time. Blocking the membrane with 5-10% nonfat dried milk in PBS for 1 hour or overnight at 4C with shaking. 4.Detecting the protein specific antibodies: Mark the position of the pre-stained markers, since they may fade away during detection. Mark the side of the membrane that was facing the gel. After the transfer, unclamp the blot sandwich and remove the sheets of blotting paper, exposing the blot membrane. Transfer conditions: (always using the Bio-Ice cooling unit) Put on the lip plug the cable into the power supply. Set speed as far as possible to keep ion distribution even. Add s standard stir bar to help maintain even buffer temperature and ion distribution in the tank. Place in tank and completely fill the rank with buffer. Repeat for the other cassette if you have one more. Lock the cassette closed with the white latch. Using a glass tube to gently roll air bubbles out)Ĩ) Close the cassette firmly, being careful not to move the gel and filter paper sandwich. (Removing any air bubbles, which may have formed it is very important for good results. Equilibrate the gel and soak the member, filter paper, and fiber pads in transfer buffer for 15 min.ġ) Place the cassette, with the gray side down, on a lean surface.Ģ) Place one pre-wetted fiber pad on the gray side of cassette.ģ) Place a sheet of filter paper on the fiber pad.Ĥ) Place the equilibrated gel on the filter paper.ĥ) Place the pre-wetted membrane on the gelĦ) Complete the sandwich by placing a piece of filter paper on the membrane. Cut the membrane and filter paper to the dimensions of the gel. (Caution: The buffer must be at 4☌ for electrophoretic transfer.) After use, return the cooling unit to the freezer for storage. Fill the Bio-Ice cooling unit with water and store it in –20C until ready to use. Maneuver the gel with forceps or gloved hands to minimize background ) ![]() (Caution: Always wear gloves when handling the gel or membrane. Transfer Buffer for approximately 10 min to remove detergent. Remove the gel from the electrophoresis apparatus and incubate it in Western Mark the orientation of the gel by cutting a corner from the bottom of the gel. Remove the glass plates from the electrophoresis apparatus and place them on a paper towel. Attach the electrophoresis apparatus to an electric power supply (the positive elecrode should be connected to the bottom buffer reservoir) Loading up 20-40ul of each sample in a predetermined order into the bottom of wells. Add Tris-glycine electrophoresis running buffer to the top and bottom reservoirs.Ģ50 mM glycine (electrophoresis grade) (pH8.3) Preparing the GEL: mount the gel in the electrophoresis apparatus. (DTT should be added just before the buffer is used, from 1M stock) Add 10-20ul of sample to same amount of 2X loading buffer (1:1), mix well, and include Protein Marker. Choose the percentage of SDS-Polyacrylamide Gel according to protein molecular size.Īcrylamide concentration (%) Linear range of separation (KD) SDS-PAGE (SDS-Polyacrylamide Gel Electrophoresis): Spin 10 min at 14K rpm -Take the supernatant into a new tube 2. Vortex well, put in to ice for 30-60 min Scrape the mixture all from slide to a 1.5 ml tube. 100-200ul of lysis buffer is added to each slide, mix tissue with pipette tips Triple-detergent lysis buffer (from Sambrook Fritsch Maniatis): (The slides can store at –80C for later study) Cutting 20 um frozen sections to a slide, 3-5 of them depends on the tissue size.
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