Title of Invention	"AN IMPROVED ISOELECTRIC FOCUSING UNIT"
Abstract	An improved isoelectric focusing unit which comprises an open ended column (1) having a side outlet (2) with a stop cock (3) in its lower end, characterized in that the said lower end is tapered and being plugged with an ion conducting polymer blend (4), a seperation solution (7), a gradient solution (8) and a cathode solution such as herein described (9) placed over the said polymer blend (4), the said tapered end containing an ion conducting polymer being placed such that it dips in an anode solution (10), a cathode (5) and an anode (6) being placed in the said cathode and anode solutions respectively.

Title of Invention

"AN IMPROVED ISOELECTRIC FOCUSING UNIT"

Abstract

An improved isoelectric focusing unit which comprises an open ended column (1) having a side outlet (2) with a stop cock (3) in its lower end, characterized in that the said lower end is tapered and being plugged with an ion conducting polymer blend (4), a seperation solution (7), a gradient solution (8) and a cathode solution such as herein described (9) placed over the said polymer blend (4), the said tapered end containing an ion conducting polymer being placed such that it dips in an anode solution (10), a cathode (5) and an anode (6) being placed in the said cathode and anode solutions respectively.

Full Text	This invention relates to An improved isoelectric focusing unit useful for I.ho separation of proteins. More particularly, this invention relates to an improved isoelectric focusing unit incorporating the ion conducting polymer blend as described and claimed in our patent application No.NF 112/95. The protein separation by isoelectric focusing has been known in the past and there are a number of units reported in literature. Some of tliese are based on liquid electrolytes while the others use gels for the separation medium. In the former case typically the protein along with special eletrolyte/ampholyte are loaded in a horizontal (or vertical) coaxial double walled glass or plastic tube having alternative coupling to drain ports which are initially sealed with tape. The high voltage ( >1500V) is applied to the the electrodes placed at the either ends of the tube for a period of 10 hrs and the fractions collected by puncturing the tape at the drain ports. If gel medium is used the same has to be cut and the protein eluted to obtain the useful fractions. Such commercially available apparatus has many drawbacks : (i) it has complex design, (ii) large quantity of sample is required ., (iii) high voltage is required to be applied and (iv) long time is required in excess of 20 hrs. for obtaining the protein fractions. Further, the exact isolectric focusing point cannot be recorded easily. There have been several modifications suggested in the past for the design of the electrofocusing unit such as J-tube, U-tube , multi nozzel etc. However, these do not lead to much improvement in the voltage or the time duration required for obtaining the isoelectric point. Further, their operation is quite combursome and different proteins cannot be easily fractionated because of the complexity of connecting tubes, electrodes etc. The main objective of the present invention is to provide an improved isoelectric focusing unit useful; I'm.- the separationof proteins . Another object is to provide an isoelctric focusing unit incorporating the ion conducting polymer blend as descrided and claimed in our copending patent application No. NF 112/95. The improved isoelctric focusing unit of the present invention which uses ion conducting polymer in a straight tube which gives isoelectric point in much shorter time, requires very little quantity of sample and the separation of the fractions is 'just by draining the tap. The apparatus, which is schematically shown in Fig.l of the drawing accompanying this specification consists of a single open ended glass column with a tapered end (1), with a side arm (2) provided with a stop cock (3) for easy collection of the protein fractions. The tapered end of the tube is plugged with the ion conducting polymer (4) above this is the separation solution (7), gradient solution (8), cathode solution (9) respectively. The tapered end containing the ion conducting polymer (4) being dipped in an anode solution'(10). A cathode (5) 'and an anode (6) being placed in the cathode and anode solutions respectively . Accordingly, the present invention provides an improved isoelectric focusing unit useful for the separation of proteins which comprises : an open"ended column (1) having side outlet (2) with a stop cock (3) in its lower portion and a tapered lower end., the lower tapered end ebing plugged with an ion conducting polymer blend (4) and bove it being placed a separation solution (7), a gradient solution (8) and a cathode solution (9) respectively, the tapered end containing the ion conducting polymer (4) being placed such that it dips in an anode solution (10), a cathode (5) and an anode (6) being,placed in the cathode and anode solution respectively. The ion conducting polymer blend used may be as described and claimed in our copending application No.NF-112/95. The separation solution used may be selected from ethylene ' glycol, glycerol, polyethylene glycol dissolved in water. The gradient solution used may be selected from a mixture of glycerol and water with carrier electrolyte, ampholyte and amphoteric long chain compounds. The cathode solution used may be selected from phosphoric acid, acetic acid or boric acid. The anode solution used may be selected from sodium hydroxide or potassium hydroxide. The main glass column (1) is straight cylinderical tube 9mm in diameter and 420 mm in length with the taper at the bottom to reduce the end diameter to 5mm which extends over 20 mm length. The side arm (2) is placed at an angle of 45 degrees to the main tube (1) and joined at 55 mm from the bottom end of the main tube and stop cock (3) is fixed to the side arm (2). The ion conducting polymer used is prepared according to the process as described in our copending patent application No.NF-112 /95 and the same is placed at the tapered end of the main tube so as to fill the same upto a length of 30mm from the bottom end . The following examples are given to illustrate the device of the present and should not be construed to limit the scope of the present invention. EXAMPLE - 1 The isoelectric focusing unit constructed as per the present invention having main glass tube of 420 mm length was operated with anode solution of 0. 1M phosphoric acid, cathode solution 0. 1M sodium hydroxide, the separation solution containing 1.5 ml glycerol in 1 ml distilled water and gradient solution of 3 ml glycerol in 2 ml water with 0.2 ml carrier ampholyte and 10 ul of α - L - arabinofuranosidase protein gave distinct separate bands placed at 7.5 cm within 4 hrs at an applied voltage of 400V and 2 mA current as compared to 20 to 24 hrs required with conventional units operated at 1500 V and 2 mA. EXAMPLE - 2 - The isoelectric focusing unit having the main glass tube of 10 cm length and 0.9 cm internal diameter was also fabricated. The other parameters were kept the same as in example 1. The application of 110 V 2 HI A current was sufficient to observe the separation of protein bands which were very closely spaced (1.0 cm) and hence could not be fractionated into pure components. However, the isoelectric focusing point could be recorded. We Claim: 1. An improved isoelectric focusing unit which comprises an open ended column (1) having a side outlet (2) with a stop cock (3) in its lower end, characterized in that the said lower end is tapered and being plugged with an ion conducting polymer blend (4), a separation solution (7), a gradient solution (8) and a cathode solution such as herein described (9) placed over the said polymer blend (4), the said tapered end containing an ion conducting polymer being placed such that it dips in an anode solution (10), a cathode (5) and an anode (6) being placed in the said cathode and anode solutions respectively. 2. A unit as claimed in claim 1 wherein the separation solution used is selected from ethylene glycol, glycerol, polyethylene glycol dissolved in water. 3. A unit as claimed in anyone of preceding claims wherein the gradient solution used is selected from a mixture of glycerol, water, carrier electrolyte and an ampholyte. 4. A unit as claimed in anyone of preceding claims wherein the cathode solution used is selected from sodium hydroxide or potassium hydroxdide. 5. A unit as claimed in anyone of preceding claims wherein the anode solution used is selected from phosophoric acid, acetic acid or boric acid. 6. An improved isoelectric focusing unit substantially as herein described with reference to the examples.

Full Text

This invention relates to An improved isoelectric focusing unit useful for I.ho separation of proteins. More particularly, this invention relates to an improved isoelectric focusing unit incorporating the ion conducting polymer blend as described and claimed in our patent application No.NF 112/95.
The protein separation by isoelectric focusing has been known in the past and there are a number of units reported in literature. Some of tliese are based on liquid electrolytes while the others use gels for the separation medium. In the former case typically the protein along with special eletrolyte/ampholyte are loaded in a horizontal (or vertical) coaxial double walled glass or plastic tube having alternative coupling to drain ports which are initially sealed with tape. The high voltage ( >1500V) is applied to the the electrodes placed at the either ends of the tube for a period of 10 hrs and the fractions collected by puncturing the tape at the drain ports. If gel medium is used the same has to be cut and the protein eluted to obtain the useful fractions. Such commercially available apparatus has many drawbacks : (i) it has complex design, (ii) large quantity of sample is required ., (iii) high voltage is required to be applied and (iv) long time is required in excess of 20 hrs. for obtaining the protein fractions. Further, the exact isolectric focusing point cannot be recorded easily.
There have been several modifications suggested in the past for the design of the electrofocusing unit such as J-tube, U-tube , multi nozzel etc. However, these do not lead to much improvement

in the voltage or the time duration required for obtaining the isoelectric point. Further, their operation is quite combursome and different proteins cannot be easily fractionated because of the complexity of connecting tubes, electrodes etc.
The main objective of the present invention is to provide an improved isoelectric focusing unit useful; I'm.- the separationof proteins . Another object is to provide an isoelctric focusing unit incorporating the ion conducting polymer blend as descrided and claimed in our copending patent application No. NF 112/95. The improved isoelctric focusing unit of the present invention which uses ion conducting polymer in a straight tube which gives isoelectric point in much shorter time, requires very little quantity of sample and the separation of the fractions is 'just by draining the tap.
The apparatus, which is schematically shown in Fig.l of the drawing accompanying this specification consists of a single open ended glass column with a tapered end (1), with a side arm (2) provided with a stop cock (3) for easy collection of the protein fractions. The tapered end of the tube is plugged with the ion conducting polymer (4) above this is the separation solution (7), gradient solution (8), cathode solution (9) respectively. The tapered end containing the ion conducting polymer (4) being dipped in an anode solution'(10). A cathode (5) 'and an anode (6) being placed in the cathode and anode solutions respectively .
Accordingly, the present invention provides an improved isoelectric focusing unit useful for the separation of proteins which comprises : an open"ended column (1) having side outlet (2) with a stop cock (3) in its lower portion and a tapered lower end., the lower tapered end ebing plugged with an ion conducting polymer blend (4) and bove it being placed a separation solution (7), a gradient solution (8) and a cathode solution (9) respectively, the tapered end containing the ion conducting polymer (4) being placed such that it dips in an anode solution (10), a cathode (5) and an anode (6) being,placed in the cathode and anode solution respectively.
The ion conducting polymer blend used may be as described and claimed in our copending application No.NF-112/95.
The separation solution used may be selected from ethylene ' glycol, glycerol, polyethylene glycol dissolved in water. The gradient solution used may be selected from a mixture of glycerol and water with carrier electrolyte, ampholyte and amphoteric long chain compounds.
The cathode solution used may be selected from phosphoric acid, acetic acid or boric acid.
The anode solution used may be selected from sodium hydroxide or potassium hydroxide.
The main glass column (1) is straight cylinderical tube 9mm in diameter and 420 mm in length with the taper at the bottom to reduce the end diameter to 5mm which extends over 20 mm length. The side arm (2) is placed at an angle of 45 degrees to the main tube (1) and joined at 55 mm from the bottom end of the main tube
and stop cock (3) is fixed to the side arm (2). The ion conducting polymer used is prepared according to the process as described in our copending patent application No.NF-112 /95 and the same is placed at the tapered end of the main tube so as to fill the same upto a length of 30mm from the bottom end .
The following examples are given to illustrate the device of the present and should not be construed to limit the scope of the present invention.
EXAMPLE - 1 The isoelectric focusing unit constructed as per the present invention having main glass tube of 420 mm length was operated with anode solution of 0. 1M phosphoric acid, cathode solution 0. 1M sodium hydroxide, the separation solution containing 1.5 ml glycerol in 1 ml distilled water and gradient solution of 3 ml glycerol in 2 ml water with 0.2 ml carrier ampholyte and 10 ul of α - L - arabinofuranosidase protein gave distinct separate bands placed at 7.5 cm within 4 hrs at an applied voltage of 400V and 2 mA current as compared to 20 to 24 hrs required with conventional units operated at 1500 V and 2 mA.
EXAMPLE - 2 - The isoelectric focusing unit having the main glass tube of 10 cm length and 0.9 cm internal diameter was also fabricated. The
other parameters were kept the same as in example 1. The application of 110 V 2 HI A current was sufficient to observe the separation of protein bands which were very closely spaced (1.0 cm) and hence could not be fractionated into pure components. However, the isoelectric focusing point could be recorded.

We Claim:
1. An improved isoelectric focusing unit which comprises an open ended column (1) having a side outlet (2) with a stop cock (3) in its lower end, characterized in that the said lower end is tapered and being plugged with an ion conducting polymer blend (4), a separation solution (7), a gradient solution (8) and a cathode solution such as herein described (9) placed over the said polymer blend (4), the said tapered end containing an ion conducting polymer being placed such that it dips in an anode solution (10), a cathode (5) and an anode (6) being placed in the said cathode and anode solutions respectively.
2. A unit as claimed in claim 1 wherein the separation solution used is selected from ethylene glycol, glycerol, polyethylene glycol dissolved in water.
3. A unit as claimed in anyone of preceding claims wherein the gradient solution used is selected from a mixture of glycerol, water, carrier electrolyte and an ampholyte.
4. A unit as claimed in anyone of preceding claims wherein the cathode solution used is selected from sodium hydroxide or potassium hydroxdide.
5. A unit as claimed in anyone of preceding claims wherein the anode solution used is selected from phosophoric acid, acetic acid or boric acid.
6. An improved isoelectric focusing unit substantially as herein described with reference to the examples.

Documents:

2456-del-1995-abstract.pdf

2456-del-1995-claims.pdf

2456-del-1995-complete specification (granted).pdf

2456-del-1995-correspondence-others.pdf

2456-del-1995-correspondence-po.pdf

2456-del-1995-description (complete).pdf

2456-del-1995-drawings.pdf

2456-del-1995-form-1.pdf

2456-del-1995-form-2.pdf

2456-del-1995-form-4.pdf

2456-del-1995-form-9.pdf

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Patent Number

193535

Indian Patent Application Number

2456/DEL/1995

PG Journal Number

31/2009

Publication Date

31-Jul-2009

Grant Date

06-Jan-2006

Date of Filing

29-Dec-1995

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	SATHIVEL CHINNATHAMBI	NATIONAL CHEMICAL LABORATORY, MAHARASHTRA, INDIA.
2	ANIL HARISHCHANDRA LACHKE	NATIONAL CHEMICAL LABORATORY, MAHARASHTRA, INDIA.
3	SUBRAMANIAM RADHAKRISHNAN	NATIONAL CHEMICAL LABORATORY, MAHARASHTRA, INDIA.

PCT International Classification Number

G03B 13/36

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA