Blot out spot in 600

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Aug 6th, 2022
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How to blot out spot in 600

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Try imaging the blot again with a shorter exposure time. This may require some optimization to get right. Your choice of membrane may give a high background. Nitrocellulose membranes generally give less background than PVDF; consider using a nitrocellulose membrane instead if high background persists.
Problem: Uneven spots on blot or speckled background A speckled or spotty background can be caused by foreign material adhering to the membrane, antibody aggregation, or dirty scanner or cassette surfaces. All are preventable by fastidious handling procedures.
Problem: Blotchy or patchy background A blotchy background can be due to a dry membrane or insufficient washing; both problems are readily solved by ensuring that the blot is immersed at all times.
Blocking is critical for clean blots. Blocking agents such as non-fat dry milk or BSA occupy non-specific binding sites resulting in lower background. Check to see if there is a preferred blocking agent and concentration for your antibody. A 1-5% blocking solution is usually recommended.
Bands appear white (if using ECL detection) Primary and secondary antibody concentration may be too high. If the antibody concentration is very high, then the substrate is consumed very quickly. This means very little light is absorbed at this point, leading to a white band when you image the blot.
Blotchy, Flecked, Or Dirty Background These artifacts are most commonly the result of uneven coating of buffer or antibody, the membrane drying out, or aggregates forming in the antibody or blocking buffer.
Problem: Uneven spots on blot or speckled background Make sure blocking reagent is fully dissolved in buffer prior to use. Make fresh buffers if making blocking buffer from dry reagents. Check premade blocking buffers for precipitates before use. Add 0.050.1% Tween 20 to blocking buffer.
To perform Western blot normalization using a single protein as a control, the blot is probed with a primary antibody specific for the protein of interest, and one directed against a normalization control. Ideally, the normalization control is a protein that is present at constant levels in every sample.

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