Use low-fluorescence PVDF membrane, and confirm uniform membrane background before you transfer. Gently agitate during every antibody incubation. Use enough reagents to prevent areas of the membrane from drying out during incubations and washes. Increase antibody volume so entire membrane surface is sufficiently covered with liquid at all times. After 3 months, the effectiveness of secondary antibodies will decrease, and background may increase. IRDye Secondary Antibodies are stable for 3 months at 4° C. Get the One-Blot Western Optimization: Using the MPX Blotting System technical note for more information. Use the MPX ™ Blotting System to optimize primary and secondary antibody dilutions. For IRDye Secondary Antibodies, a good starting dilution is 1:20,000. Excess Tween 20 (0.5 – 1%) may decrease signal.ĭo not add secondary antibodies at concentrations higher than 1:5,000, as high background will result. Make sure that 0.1 – 0.2% Tween 20 is present in buffer. If background persists, increase the number of washes and buffer volume. With IRDye ® 680LT Secondary Antibodies, use SDS (0.01 – 0.02% final concentration) and Tween 20 (0.1 – 0.2% final concentration) during the detection incubation step. Lack of detergentĪdd Tween ® 20 to primary, secondary, and wash steps at 0.1 – 0.2%.įor PVDF membranes, add SDS to secondary antibody incubation (not primary antibody incubation or wash step) at 0.01 – 0.02%.ĭo not add detergent to the blocking step. Instead, try an Intercept ® Blocking Buffer. Optimize to determine the best blocking buffer for your primary antibody Cross-reactivity of antibody with blocker, especially milkĭon’t use milk for blocking, as milk typically contains IgGs that cross-react with anti-goat secondary antibodies. Blocking conditionsĭifferent primary antibodies will react differently in different blocking buffers. Before you use them, clean dishes, bags, or trays for incubations with methanol. Always handle membranes carefully and with clean forceps.

Western blot ladder but no protein