Techniques – Wild, Wild West(erns)

Need some help to tame that wild western blot? Here are some tips and tricks to help you along the way –

  • Always check ladder migration pattern based on specific gel and electrophoresis conditions, as these factors can shift the apparent molecular weights from the supposed “standard” ladder image given out by the company.
  • When testing a new antibody, leave the blot intact, opting to strip it and perform a control protein blot after probing for your target protein. Cutting the blot and using different pieces for your target and control protein on a first try may obscure alternative target protein isoforms or off-target background staining.
  • High background? Try a more stringent blocking condition than just BSA or milk by adding goat serum or fish gelatin to your solution. Blocking overnight also can help clean up your blots.
  • Is you gel “smiling” or “frowning”? This usually happens when your sample buffer has too much salt.
  • Make sure your PVDF membrane is pre-activated with methanol for 20 minutes before making gel sandwich. It’s also a good idea to mark which side of the membrane is facing the gel with a sharpie, on a top corner.
  • It’s always a good idea to do a Ponceau stain on your membrane after transfer to make sure your transfer went all right. Alternatively, you can also stain your gel with Coomassie blue.
  • It is possible to over-transfer, especially for low MW proteins (<10 kDa) – optimize transfer time or reduce voltage. On the other hand, high MW proteins will take longer time to transfer.
  • Have a dirty secondary? Consider adding a wee-bit of Tween-20 to your washes (0.01-0.5%).
  • When loading your samples, press on the pipet just enough to get any possible air bubbles out and run the tip through the running buffer in the tank before putting it in the well. And make sure your sample was denatured prior to running.
  • If power supply reading shows 0 when switched on, make sure your power cables are properly connected to the power supply. If that doesn’t work, check for broken electrodes or blown fuses. Lastly, try with a higher limit power supply.
  • As always, make sure you write down the protocol before-hand and check through every step when performing. This will help you track your steps back to see at which step things could have gone wrong.

 

Source - http://prooffreaderplus.blogspot.com/2014/05/one-of-my-favourite-memories-good-bad.html
Source – http://prooffreaderplus.blogspot.com/2014/05/one-of-my-favourite-memories-good-bad.html

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