WB Technical Resources

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Troubleshooting guides

Troubleshooting guides

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Western Blot Troubleshooting: Distorted Bands

Distorted bands can make it very hard to interpret your results. Common distortions include smile shaped bands with the edges trailing upward, diffuse bands that are broad or blurry, and streaked bands that trail off in several directions. Make sure your next blot has even, crisp bands by following these tips:

Problem Cause Solution
Curved, "smiling" bands Electrophoresis voltage too high Reduce voltage; run the gel slower for more consistent results
Overheated gel Reduce voltage or run the gel on ice or in a cold room (-4C)
Streaked or diffuse bands Incomplete contact between gel and membrane during transfer Use thicker filter paper in sandwich
Squeeze air bubbles and excess buffer from between membrane and gel
Slippage of membrane during transfer Avoid agitating or moving the gel or membrane during transfer
Blurry bands Electrophoresis voltage too high Run the gel for longer at a lower voltage
Impropher loading buffer composition Mix new loading buffer, or use Boster formulated loading buffer
Air bubbles trapped between gel and membrane during transfer Carefully remove bubbles by squeezing them out with a sterile glass rod
Ghost bands Overexposure during visualization Decrease the exposure time
Loading sample too concentrated Reduce the amount of sample loaded. See Boster's Western blot troubleshooting guide for load optimization tips
Antibody concentration too high Dilute your antibody solutions using Boster's antibody diluent medium
Blot was moved during transfer Avoid agitating or moving the gel or membrane during transfer
Uneven, crooked bands Poor gel polymerization Check gel concentration and ensure complete dissolution of SDS before casting gels
Varying salt concentration between wells Make sure the salt concentration in different samples is similar

Keywords: Western Blot, Troubleshooting, Distorted, Streaked, Blurry, Smile Bands, Ghost Bands

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