![]() ![]() 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. Conditioned media is collected in 15 mL tubes, spun down at 5000 rpm for 5 minutes to form a pellet.ġ) Cells can be treated with serum free (SF) conditions by washing with 1x PBS and adding 4-7 mL DMEM with 0.7% FBS or BSA.Ģ) 3T3-L1 cells at Day 0 should not be kept under these conditions for more than 6 hours, as cells begin to die at a rapid rate without serum.ģ) Spin down the media collected from these cells since there will be cells suspended in it that must be cleared before proceeding.Ĥ) Samples are boiled on a 95 ☌ heat block for 5 minutes (preadipocytes should be boiled for 15 minutes to fully disperse blob)ĥ) Pipet the sample to fully disperse the blob and remove an aliquot to use as a working sample.Ħ) 4x SDS loading buffer is added to the working sample to a concentration of 1x.ħ) Samples are boiled again at 95 ☌ for 5 minutes to denature and allow for proper dispersal of the blob.Ĩ) Working and stock samples should be stored at -20 ☌.Cells from 6 well plates should be lysed in 750 μL to 1 mL of WLB with protease tablets. Cells from 10 cm plates are are collected at 100% confluence, Day 0, in 1 mL of Western Lysis Buffer (WLB) and stored at -20 ̊C.Sample Preparation: Cell Lysate and Conditioned Media ![]()
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