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Study Notes: Achieving Good Separations with SDS-PAGE

The keys to achieving good separations with SDS-PAGE fall into four distinct categories as follows.

  • Absolute cleanliness of the apparatus especially the glass plates.
  • Using high purity reagents for all aspects of gel and sample preparation.
  • Knowing how to assemble and use the apparatus.
  • Hands-on experience at running SDS-PAGE.

Cleanliness of the apparatus and the glass plates is crucial to producing a gel with clear and distinct bands. After each run the apparatus should be cleaned and dried as follows.

  • Glass plates are precision engineered (and hence expensive) and very fragile around the edges - treat with great care as you clean.
  • Wash with hot water using a powdered alkaline detergent such as ‘Pyroneg’.
  • Rinse all detergent and suds off with plenty of running hot water.
  • Wash off the hot water with plenty of running cold water.
  • Wash off the cold water with plenty of high quality distilled or deionised water.
  • Stand glass plates vertically to dry.
  • Dry in a 37°C oven.
  • Store covered and away from dust.
  • Examine plates for any streaks, prior to use.

Reagents used for sample preparation and gel preparation should be high quality laboratory reagents. Reagents for the tank running buffer does not need to be of such high quality and a general grade of laboratory reagent will suffice.

The following general rules will be useful.

  • Be very careful with the shelf life of reagents.
  • Store at the required temperature.
  • Watch for precipitation of reagents such as SDS in the fridge.
  • Always check reagents for microbial contamination before use.
  • Discard single use preparations once used.
  • Treat acrylamide and powdered SDS with great respect.

The use of apparatus is beyond the scope of these Study Notes but you should keep the following general rules in mind when assembling the apparatus.

  • Read and follow all instructions carefully.
  • Watch someone assemble the apparatus first.
  • Assemble under the guidance of experienced staff the first few times.
  • Do not force any part of the apparatus to make it fit.
  • Avoid getting fingerprints on the inner side of the glass plates.
  • Fill the glass plates carefully to avoid bubble formation.
  • Check for leaks after filling - a leak will lead to distortions in protein bands.
  • If it leaks - start again!
  • Fill the sample wells slowly and carefully to avoid sample overflowing from one
    well to another.
  • When the apparatus is connected to the electric current it is a potential
    electrocution hazard - treat with caution.
  • Check from time to time to see that the protein samples are moving into the
    stacking gel - no movement means a problem.
  • Check to ensure that the dye front does not run off the end of the gel.

Hands-on experience can only be gained one way! We recommend that you gain this experience as soon as possible.

One final point
How long should a gel be run for? Can I leave it running while I go to lunch or the library?

The short answer to the second question is yes as long as you know the answer to the first question. A standard 15 cm x 15 cm SDS-PAGE gel should be run for approximately 120 to 150 mA hours.

This means that if you want to run a gel overnight from say 5.00 pm to 9.00 am the next day) it would be running for 16 hours.

120 to 150 mA hours    

= 7.5 to 9.4 mA
16 hours

So the powerpack would be set to a constant current of say 8 mA. Always be a little conservative so the gel does not run off before you get in to turn it off!

Remember that many SDS-PAGE set-ups allow one or two gels to be run at once. If two gels are being run, then 240 to 300 mA hours are required and in the example above, the current selected would be 16 mA.

One word of caution
Running gels at high current to get the gel to run quickly before knock off time leads to the overheating of the gel. As the edges of the gel are able to dissipate the heat to the tank buffer more effectively than the middle of the gel the proteins on the edges migrate more slowly that those in the middle of the gel.

The result is proteins and the dye front that bend down in a ‘smile’. This is not a good look on a gel!

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