...
| Expand | ||
|---|---|---|
| ||
Lysis buffer (from Abcam) pH 8.0 150 mM NaCl 10% NP40 or Triton X-100 50 mM Tris
Running buffer (from Bio-Rad) pH 8.3 25 mM Tris 192 mM glycine 0.1% SDS
Gel stain (from Abcam) 0.3 M CuCl2 Gel destaining buffer (from Abcam) pH 8.0 0.25 M Tris 0.25 M EDTA
Transfer buffer (from Abcam) 48 mM Tris 39 mM glycine 0.04% SDS 20% methanol
Membrane stain (Ponceau Red) - stock (from Abcam) Dilute 1:10 for washing the membrane 2% Ponceau S 30% trichloroacetic acid 30% sulfosalicylic acid
TBST (Antibody/BSA buffer) (from Abcam) 100 mL TBS 10x (pH 7.6)
900 mL dH2O 1 mL Tween20
Block in 5% BSA (5g BSA in 100 mL TBST)
|
| Expand | ||||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| ||||||||||||||||||||||||||||||||||||||||||
Results of BCA Protein Assay A BCA Protein Assay was run to determine the concentration of protein in our samples. In the assay, protein samples were analyzed in two dilutions: 75uL sample + 75uL dH2O and 25uL sample + 125 uL dH2O. These were compared to BSA protein standards. 150uL of each dilution of sample or BSA was added to 150uL of BCA working reagent (25 A: 24B: 1C) in a microwell plate and scanned using the Tecan plate reader at 562nm absorbance. A standard curve was created, and absorbances of the samples were compared against this curve to produce predicted protein concentrations.
See attached document for details of experimental results. In this iteration of the Western blot, 10uL of undiluted protein sample was added to 10uL of sample buffer, and 16uL of this solution was added to each well. Based on the concentrations inferred from the BCA assay and our protocol for loading the gel wells, the calculated amount of protein added to each well was:
|
...