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Want to save on reagents? |
 You can reduce the amount of your ladder (the molecular weight standard) you use by a half to a third of the recommended amount and still get a clear transfer. |
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Combine your protein ladders |
 If you use two different types of ladders when you run your protein gel, you end up with less wells to load your samples. So try adding both ladders into the same well to get an extra well to load samples in. |
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 When loading your sample into your protein gel, you may need to load larger volumes than what
your wells can hold.
Try this: 1) carefully load half the sample 2) run the gel
slowly at a low voltage to start stacking that half into the gel then 3) load
the remaining half. |
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When loading sample into wells for protein gels, don’t
push the pipette button to its ‘second’ stop – it makes air bubbles. To get more control when loading wells, push the pipette button only to the ‘first stop’, then place your finger onto the top
of the pipette tip and your sample will slowly eject itself. Then you can easily remove the tip before air bubbles get loaded into the well. 
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If your bands are running unevenly (your gel ‘smiles’ at you) your migration could be either too fast or the buffer is getting too hot. Consider reducing voltage or placing the gel apparatus in a bucket of ice. |
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An easy way to ensure a better blot |
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Using PVDF membranes to blot? PVDF membranes tend to blot best on the top side (the side which faces you when you roll the PVDF out), so make sure all your membranes are the same side up and preferably with the top side in contact with the gel.
Yes this happens even though the manufacturers claim that PVDF blotting membranes have identical sides!
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If you use ECL detection reagents to visualise your blots photographically on a film, you can see your bands clearly. But not your blot, or some ladders. This can make it harder to judge your bands later.
To mark out the parameters of your blot, such as it's corners or the bands of your ladder, try poking the blot with a needle. Do this when you have just finished transferring the proteins from your gel to your blot, before you split apart the blot from the gel. These poked marks will show through onto your film later. |
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To use less than 2 ml of diluted antibodies per blot
pack the blot tightly into a sealy-bag, and add your antibodies onto the
protein side. |
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Save time between antibodies |
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Instead of stripping your blot (which may be a hassle),
add 0.02%
sodium azide to the wash buffer to quench the chemiluminescent
signal from your first antibody, before adding the antibody for your next
protein to detect. This only works if
the next antibody to be added is derived from a different animal from your
first (e.g. a rabbit-derived antibody followed by a mouse-derived antibody). 
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Do you visualise your blots photographically on a film? |
 Want to mark out your blot clearly? If you use ECL detection reagent to visualise your blots photographically on a film, you can see your bands clearly - but not your blot, or some ladders. This can make it harder to judge your bands later. To mark out the parameters of your blot, such as its corners or the bands of your ladder, try poking the blot with a needle. Do this when you have just finished transferring the proteins from your gel to your blot, before you split apart the blot from the gel. These poked marks will show through onto your film later. |
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