Pre-staining protocol for a protein sample with a pyrylium dye and respective kit
The invention relates to a protocol for pre-staining a protein prior to electrophoresis, an electrophoresis method including the protocol, and a kit for carrying out the protocol. The protocol comprises the steps of incubating the protein sample with a pyrylium dye in the presence of a buffer, a detergent and a denaturing agent.
1. A method for pre-staining a protein sample prior to electrophoresis, comprising reacting the protein sample with a pyrylium dye in the presence of a buffer, a detergent and a denaturing agent to produce a labeled protein sample.
2. The method according to claim 1 wherein the dye has a low quantum yield when unconjugated and a high quantum yield when conjugated.
3. The method according to claim 1 , wherein the pyrylium dye is a non-sterically hindered pyrylium dye.
4. The method according to claim 2 , wherein the detergent comprises SDS.
5. The method according to claim 1 , wherein the denaturing agent comprises one or more of DTT, tributyl-phosphine (TBP) and tris(2-carboxyethyl)phosphine (TCEP).
6. The method according to claim 1 , wherein incubation is carried out at a pH from 9 to 10.
7. The method according to claim 5 , wherein the incubation is carried out at about pH 9.3.
8. The method according to claim 1 , wherein incubation is carried out at a temperature of from 70 to 95° C. and for from 1 to 60 minutes.
9. The method according to claim 8 , wherein incubation is carried out at about 75° C. for about 7 minutes.
10. The method according to claim 1 , wherein the buffer includes a sulphonate group.
11. The method according to claim 10 , wherein the buffer comprises one or more of CAPSO, CHES, TAPS, TES and HEPES.
12. A gel-electrophoresis method, the method comprising the steps of pre-staining a protein sample using a protocol according to claim 1 and electrophoresing the sample on a gel.
13. The method according to claim 12 , wherein the gel is a cross-linked polyacrylamide gel.
14. A kit for performing a protein pre-stain protocol, the kit comprising a mixture comprising a pyrylium dye, a detergent and a denaturing agent.
15. The kit according claim 14 , wherein the detergent comprises SDS.
16. The kit according to claim 14 , wherein the denaturing agent comprises one or more of DTT, tributyl-phosphine (TBP) and tris(2-carboxyethyl)phosphine (TCEP).
17. The kit according to claim 14 , further comprising a buffer that includes a sulphonate group.
18. The kit according to claim 17 , wherein the buffer comprises one or more of CAPSO, CHES, TAPS, TES and HEPES.
19. The kit according to claim 14 , further comprising an electrophoresis vessel, the vessel including a prepackaged electrophoresis gel.
20. The kit according to claim 19 , the electrophoresis vessel being an integrally formed plastics strip or continuous tape, having one or a plurality of electrophoresis volumes formed therein, each volume including a prepackaged electrophoresis gel.