9. Load 5 μL of the polysaccharide marker or any other suitable
marker for your application.
10. Run the gel at 400 V and 4
C (see Note 21).
11. Do not touch the gel from now on. Touching the gel might
cause marks that will be stained.
12. Stop electrophoresis. Carefully remove gel cassette and gel.
13. Incubate gel for at least 1 h in freshly prepared fixing solution.
14. Incubate gel for 30 min in alcian blue staining solution. The
alcian blue solution can be reused for staining up to ten gels.
15. To remove remaining alcian blue, wash the gel in water. Repeat
this step three to four times. Leaving the gel in water overnight
might reduce unwanted background staining.
16. Remove the water and gently shake the gel in oxidizing solution for 5 min.
17. Wash three times for 15 min with 500 mL of water.
18. Incubate for 10 min in silver nitrate solution.
19. Wash three times for 10 min with water to minimize the
aggregation of insoluble silver salts.
20. Gently shake the gel in 100 mL formaldehyde solution. Carefully observe the staining progress and stop the staining reaction with stop solution when signal intensities are sufficient.
21. The gel can be stored in water or dried after incubation for at
least 4 h in drying solution.
4 Notes
1. We recommend using an incubator instead of a heating block
to prevent the condensation of water inside the tube lid.
2. Capsule polymerases are cytosolic enzymes and DTT is added
to mimic the cytosolic reducing environment.
3. Nucleotide activated substrates are freeze-dried with different
counter ions depending on the method of preparation and the
supplier. Dissolve the substrates according to the molecular
weight given by the supplier. We recommend to either weigh
in amounts >5 mg or dissolve the entire batch of material to
get precise results.
4. Cps4B consists of two domains. The N-terminal domain
adopts a glycosyltransferase A (GT-A) fold and the C-terminal
domain adopts a phosphotransferase fold similar to the glycosyltransferase B (GT-B) fold (details regarding the domain
organization, cloning, and purification of Cps4B and Cps11D
and the purification and NMR analysis of their reaction
324
Christa Litschko et al.
marker for your application.
10. Run the gel at 400 V and 4
C (see Note 21).
11. Do not touch the gel from now on. Touching the gel might
cause marks that will be stained.
12. Stop electrophoresis. Carefully remove gel cassette and gel.
13. Incubate gel for at least 1 h in freshly prepared fixing solution.
14. Incubate gel for 30 min in alcian blue staining solution. The
alcian blue solution can be reused for staining up to ten gels.
15. To remove remaining alcian blue, wash the gel in water. Repeat
this step three to four times. Leaving the gel in water overnight
might reduce unwanted background staining.
16. Remove the water and gently shake the gel in oxidizing solution for 5 min.
17. Wash three times for 15 min with 500 mL of water.
18. Incubate for 10 min in silver nitrate solution.
19. Wash three times for 10 min with water to minimize the
aggregation of insoluble silver salts.
20. Gently shake the gel in 100 mL formaldehyde solution. Carefully observe the staining progress and stop the staining reaction with stop solution when signal intensities are sufficient.
21. The gel can be stored in water or dried after incubation for at
least 4 h in drying solution.
4 Notes
1. We recommend using an incubator instead of a heating block
to prevent the condensation of water inside the tube lid.
2. Capsule polymerases are cytosolic enzymes and DTT is added
to mimic the cytosolic reducing environment.
3. Nucleotide activated substrates are freeze-dried with different
counter ions depending on the method of preparation and the
supplier. Dissolve the substrates according to the molecular
weight given by the supplier. We recommend to either weigh
in amounts >5 mg or dissolve the entire batch of material to
get precise results.
4. Cps4B consists of two domains. The N-terminal domain
adopts a glycosyltransferase A (GT-A) fold and the C-terminal
domain adopts a phosphotransferase fold similar to the glycosyltransferase B (GT-B) fold (details regarding the domain
organization, cloning, and purification of Cps4B and Cps11D
and the purification and NMR analysis of their reaction
324
Christa Litschko et al.