Pouring IEF gels (old fashion method):

1. Clean plates with RBS/ethanol and place in standard minigel pourer.
2. prepare the gel solution

   2 thin gels		2 thick gels
   6.19 g urea (electrophoresis grade		16.48 g urea
   4.5 ml water		12 ml water
   1.5 ml acrylamide (30:0.8)		4ml acrylamide
   300 µL 3-10 ampholines*		800µL 3-10 ampholines*
   300 µL 4.5-6 ampholines*		800µL 4.5-6 ampholines*

   
   * adjust ratio and range of ampholines for pI of target proteins.
3. dissolve urea and degas for 2 to 3 minutes.
4. add 225µL / 600µL NP-40 - pipeting both ampholines and NP40 is easier if you cut off the pipet tip with a pair of scissors.
5. Filter solution through a syringe (0.22µm) filter.
6. add 37.5µL / 100 µL10% ammonium persulfate and the 3.75µL / 10µLTEMED. This starts polymerization, so once added you have to keep moving.
7. Pour gels – and let polymerize for 2 hours
8. RUN with basic buffer in upper resovior and acidic buffer in lower. (use NaOH and Phosphoric acid buffers - degas NaOH buffer before using)
9. RUN at 100V for 30 minutes, 200V for 1 hour, 400 V overnight, 600V for 30 min.
   

Sample preparation:

IEF samples are dissolved in urea/NP40 or urea/CHAPS buffer. This buffer is made up and stored at -20°C as a common stock. Take an aliquot and use it, discard what you do not use. DO NOT REFREEZE.

Solubilize an IP in 25-50 µL IEF sample buffer. Incubate samples at 60ºC for 5 min. and then microfuged for 5 min at room temperature prior to loading.

Upper buffer (basic) lysine/arginine buffer
Lower buffer (acidic) phosphoric acid buffer

load samples (and standards). Run following biorad recommendations
1 hour at 100 V
1 hour at 250 V
1 hour at 500 V

Blot gel onto nitrocellulose with 0.1% SDS in buffer