1/8/2024 0 Comments Western blot protocol10x TTBS is a stock solution in the cold room. Alternatively the transfer can run at a higher voltage for a few hours, but an ice block must be placed in the transfer apparatus to prevent overheating.ġ. Place the lid on and run at 35 volts overnight. Pour the 1x transfer buffer in the tray into the transfer apparatus and add any additional buffer needed to fill the apparatus to the top. Proteins are negatively charged because of the SDS and will migrate to the positive (red) charge.Ĩ. Place transfer clip in transfer apparatus so that the black side of the clip is facing the red side of the apparatus. Add another piece of Whatman paper, then the other sponge and close transfer clip.ħ. Prewet the membrane in the transfer buffer for a few seconds then place the membrane on top of the gel.Ħ. Place a sponge down on the clear side of the clip, followed by two pieces of Whatman paper, then the gel.ĥ. Disassemble gel apparatus using a prying knife and trim off the stacking gel and the bottom part that has curled up.Ĥ. Prepare 3 pieces of Whatman paper cut to the size of the gel and one membrane cut a bit smaller for each gel.ģ. Fill a tray with 1x Transfer Buffer and place a transfer clip in the tray with the clear side down.Ģ. Place the samples and run gel at 125 volts (constant) for ~90 minutes or until blue dye runs to the bottom of the gel.ġ. Once gel is firmly in place add running buffer to the center of the gel apparatus, between the gels, check for any leaks, then add Buffer to the outside of the gels about ½ or 2/3 the way up the apparatus.ģ. Select performed gels based on the number of wells needed and set up the gel apparatus.Ģ. Remove 2 μl of the sample and add it to the 1 ml of protein assay solution and proceed with Bradford analysis.ġ. Spin samples down for 1-2 minutes to cool them down and collect the sample that has condensed on the lid of the tube.Ĥ. ![]() Preheat the stock sample at 60- 80 ̊C for 1-5 minutes.Ģ. If the stock sample is being used, use the following procedure to determine protein concentration:ġ. If concentration is determined from the working sample, remove 2 μl of the sample after the blob has been fully dispersed and add it to 1 ml of protein assay solution and proceed with the Bradford analysis. ![]() **Protein concentration can be obtained from the working sample immediately before addition if loading buffer (preferred) or subsequently from the stock sample.
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