10 Ways to Screw Up a Western Blot

After a few months in the wet lab, I have become extremely proficient at messing up a western blot. Here are my top 10 tips so you can screw up too!

  1. Follow the wrong set of instructions for making acrylamide gel.
  2. Follow the correct set of instructions for making acrylamide gel. Make only half as much as you need.
  3. Fill the resolving gel too high. Be unable to insert stacking gel combs.
  4. Follow the correct set of instructions to make acrylamide gel. Prepare the correct amount of acrylamide gel. Fill the resolving gel to the correct height. Realise you’ve used combs which were too thin and there is now acrylamide everywhere causing your samples to dangle weirdly in the wells.
  5. Prepare samples with the wrong buffer. Twice.
  6. Prepare new samples. Throw them in the bin instead of the old ones.
  7. Forget that you have to prepare transfer buffer and quickly whip it together. Find buffer doesn’t fill the tank to the required level for transferring. Do not have enough time to produce transfer buffer again. Panic and assume it will be fine to top it up with distilled water. Realise that it was originally the perfect amount of buffer and would reach the correct level once an ice pack is added. Have tank overflow.
  8. Correctly prepare and run gels. Transfer proteins onto filter paper.
  9. Mistake primary and secondary antibodies. Discover after an overnight incubation.
  10. Wash out primary antibodies. Throw membrane into the sink.

And, hopefully, #11 won’t be “Have supervisor discover this post”.

Craig Burns | Cross post from craigburns.co.uk

One clap, two clap, three clap, forty?

By clapping more or less, you can signal to us which stories really stand out.