Rapid Screening of the Epidermal Growth Factor Receptor Phosphosignaling Pathway via Microplate-Based Dot Blot Assays
Figure 4
Impact of lysis buffer components on dot blot performance—Detergents. Lysates (pink bars) or buffers alone (blue) were diluted in PBS, spiked with 100 ng GAPDH and assayed for changes in GAPDH detection. The green bars show detection of the native GAPDH present in A431 lysates (no GAPDH was spiked into these samples). The CPS signal for GAPDH in PBS is displayed by the orange Bar. Each bar represents the average of 3 individual replicates. Protein concentrations for the lysates used were determined to be (a) nondetergent buffer, 494 ng/μL, and (b) RIPA buffer, 2443 ng/μL. The RIPA buffer was diluted to 494 ng/μL prior to setup. Total protein lysate loaded was as follows: 0.2% = 49 ng; 1% = 295 ng; 2% = 494 ng; 10% = 2950; 20% = 4940 ng; 50% = 12350 ng.
