VWR shiroGEL Vertical 20 Manual De Instrucciones página 22

10. Carefully insert the comb making sure that no air bubbles get trapped under the
ends of the comb teeth as these will inhibit sample progression.
11. Allow the stacking gel polymerize for 30 minutes.
For gels without stacking layers:
1. Add 70µl of TEMED for shiroGEL VERTICAL 20
generating air bubbles.
2. Fill the glass plates again avoiding generating any air bubbles. Filling must be
performed quickly before the TEMED causes the gel to become too viscous.
3. Carefully insert the comb making sure that no air bubbles get trapped under the
ends of the comb teeth as these will inhibit sample progression.
4. Let the gel polymerize. Usually this takes around 15 minutes but this can vary due
to the freshness of the reagents used. If polymerization is taken a lot longer than
this, use fresher stock solutions or add more APS and TEMED.
Sample Preparation & Loading
Preparation of denatured protein samples for loading:
The instructions given below are for denatured samples. For Native samples, please
consult a laboratory handbook.
1. Prepare the protein samples for loading. The volume of sample depends on the
capacity of the wells (See Comb specifications page 25).
2. Using a 0.5 ml micro-centrifuge tube or other convenient receptacle, combine the
protein sample and 4 X sample buffer. It is always advisable to use protein
markers in one of the end lanes to indicate sizes of bands. These should be
prepared according to the manufacturer's instructions.
3. Heat the samples in a water bath or heating block for 2 minutes to denature the
samples.
4. Centrifuge the samples in a micro-centrifuge for 20 seconds at 12,000 rpm. The
protein samples are now ready to load.
gels and mix well but avoid
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