US4559120A — Agarose gel electrophoresis technique for the det…

Claim 1

Independent Claim 210 words

An electrophoretic method of detecing human isoamylases comprising providing an aqueous buffer having a pH of between about 8.4 and about 9.2, said buffer consisting essentially of water, barbital anion at a concentration of between about 0.04 M and about 0.08 M, tris cation at a concentration of between about 0.03 M and about 0.07 M, sodium cation at a concentration of between about 0.03 M and about 0.07 M, said tris cation and said sodium cation being present in molar ratios of between about 0.5:1 and about 2:1, incorporating said buffer in an agarose gel of between about 0.4 and about 1.5 weight percent agarose, applying biological samples to said gel, subjecting said gel to a direct current electrical potential of between about 50 and about 400 volts and placing said gel in heat exchange relationship with a cooling block maintained at a temperature of between about 2° C. and about 25° C. so as not to denature the isoamylases or destabilize the gel, and developing said gel to reveal isoamylase bands, said method providing a clear separation of P 1 , P 1b , P 2 , P 3 , P 4 , S 1 , S 2 , S 3 , S 4 and S 5 bands.

Claim 2

Dependent Claim 210 words

An electrophoretic method of detecing human isoamylases comprising providing an aqueous buffer having a pH of between about 8.4 and about 9.2, said buffer consisting essentially of water, barbital anion at a concentration of between about 0.04 M and about 0.08 M, tris cation at a concentration of between about 0.03 M and about 0.07 M, sodium cation at a concentration of between about 0.03 M and about 0.07 M, said tris cation and said sodium cation being present in molar ratios of between about 0.5:1 and about 2:1, incorporating said buffer in an agarose gel of between about 0.4 and about 1.5 weight percent agarose, applying biological samples to said gel, subjecting said gel to a direct current electrical potential of between about 50 and about 400 volts and placing said gel in heat exchange relationship with a cooling block maintained at a temperature of between about 2° C. and about 25° C. so as not to denature the isoamylases or destabilize the gel, and developing said gel to reveal isoamylase bands, said method providing a clear separation of P 1 , P 1b , P 2 , P 3 , P 4 , S 1 , S 2 , S 3 , S 4 and S 5 bands.

Claim 3

Dependent Claim 16 words

A method according to claim 1 including measuring the relative intensities of said developed isoamylase bands.

Claim 4

Dependent Claim 32 words

A method according to claim 1 wherein said developed isoamylase bands are measured by drying the gel and subsequently measuring the optical transmission of the gel in the region of the bands.

Claim 5

Dependent Claim 19 words

A method according to claim 1 wherein the pH of said buffer is between about 8.7 and about 8.9.

Claim 6

Dependent Claim 45 words

A method according to claim 1 wherein said gel is less than about 1 mm thick and each of said biological samples is applied in amounts providing amylase at levels of between about 5×10 -4 and about 4×10 -2 IU per cm of band length.

Claim 7

Dependent Claim 19 words

A method according to claim 1, said buffer containing calcium cation up to a concentration of about 0.002 M.

Claim 8

Dependent Claim 19 words

A method according to claim 1, said buffer containing lactate anion up to a concentration of about 0.002 M.

Claim 9

Dependent Claim 118 words

An electrophoresis plate for separating human isoamylases comprising a backing sheet and a gel on one surface of said sheet, said gel including an aqueous buffer with a pH of between about 8.4 and about 9.2, and consisting essentially of water, barbital anion at a concentration of between about 0.04 M and about 0.08 M, tris cation at a concentration of between about 0.03 M and about 0.07 M, sodium cation at a concentration of between about 0.03 M and about 0.07 M, said tris cation and said sodium cation being present in molar ratios of between about 0.5:1 and about 2:1, and agarose in amounts of between about 0.4 and about 1.5 weight percent of said gel.

Claim 10

Dependent Claim 118 words

An electrophoresis plate for separating human isoamylases comprising a backing sheet and a gel on one surface of said sheet, said gel including an aqueous buffer with a pH of between about 8.4 and about 9.2, and consisting essentially of water, barbital anion at a concentration of between about 0.04 M and about 0.08 M, tris cation at a concentration of between about 0.03 M and about 0.07 M, sodium cation at a concentration of between about 0.03 M and about 0.07 M, said tris cation and said sodium cation being present in molar ratios of between about 0.5:1 and about 2:1, and agarose in amounts of between about 0.4 and about 1.5 weight percent of said gel.

Claim 11

Dependent Claim 19 words

A plate according to claim 8, said buffer containing calcium cation up to a concentration of about 0.002 M.

Claim 12

Dependent Claim 19 words

A plate according to claim 8, said buffer containing lactate anion up to a concentration of about 0.002 M.

Claim 13

Dependent Claim 19 words

A plate according to claim 8, wherein the pH of said buffer is between about 8.7 and about 8.9.

Claim 14

Dependent Claim 141 words

A prepackaged kit for effecting electrophoretic separation of human isoamylases comprising a dry mixture of tris barbital, barbital and sodium barbital which when added to a given volume of water provides an aqueous buffer with a pH of between about 8.4 and about 9.2, and said buffer consisting essentially of water, barbital anion at a concentration of between about 0.03 M and about 0.08 M, tris cation at a concentration of between about 0.03 M and about 0.07 M, sodium cation at a concentration of between about 0.03 M and about 0.07 M, said tris cation and said sodium cation being present in molar ratios of between about 0.5:1 and about 2:1, and a prepared agarose gel consisting of agarose at a concentration of between about 0.4 and about 1.5 weight percent and buffer of like composition to said aqueous buffer.

Claim 15

Dependent Claim 141 words

A prepackaged kit for effecting electrophoretic separation of human isoamylases comprising a dry mixture of tris barbital, barbital and sodium barbital which when added to a given volume of water provides an aqueous buffer with a pH of between about 8.4 and about 9.2, and said buffer consisting essentially of water, barbital anion at a concentration of between about 0.03 M and about 0.08 M, tris cation at a concentration of between about 0.03 M and about 0.07 M, sodium cation at a concentration of between about 0.03 M and about 0.07 M, said tris cation and said sodium cation being present in molar ratios of between about 0.5:1 and about 2:1, and a prepared agarose gel consisting of agarose at a concentration of between about 0.4 and about 1.5 weight percent and buffer of like composition to said aqueous buffer.

Claim 16

Dependent Claim 29 words

A kit according to claim 12 wherein said dry mixture includes calcium lactate so that said aqueous buffer contains calcium lactate up to a concentration of about 0.002 M.

Claim 17

Dependent Claim 20 words

A kit according to claim 12 wherein the pH of said aqueous buffer is between about 8.7 and about 8.9.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1988-12-16	1989-06-17	1989-12-17	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1992-12-16	1993-06-17	1993-12-17	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	1996-12-16	1997-06-17	1997-12-17	$7700.00	Window Closed / Lapsed

Agarose gel electrophoresis technique for the determination of amylase isoenzymes

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