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6 native anionic polyacrylamide gel recipe in austria

  • Native Polyacrylamide Gel Electrophoresis - an

    Native polyacrylamide gel electrophoresis is performed using 6% acrylamide gels in Tris‐boric‐EDTA (8.9 mM Tris base, 8.9 mM boric acid, 0.2 mM Na 2 EDTA) buffer, pH 8, as described by Laemmli (1970). Staining for GSNOR activity is carried out using a modification of the method reported by Seymour and Lazarus (1989) and Fernández et al (2003).

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  • Introduction to Polyacrylamide Gels | LSR | Bio-Rad

    Peel the small piece of gel containing the DNA from the plastic wrap. 3. Transfer the gel slice to a microfuge tube. 4. Crush the gel slice against the wall of the microfuge tube with the disposable pipette tip. 5. Add 1 - 2 volumes of elution buffer to the gel slice (e.g., if the estimated gel slice volume is …

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  • Section VII: Separation of DNA in Polyacrylamide Gels

    2021-7-8 · Optimal separation of the thylakoid membrane protein complexes by lpBN-. PAGE, in turn, was obtained by using acrylamide gradient of 3.5-12.5% (w/v) T, 3% (w/v) C in the separation gel and 3% (w/v) T, 20% (w/v) C in the stacking gel. Importantly, the …

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  • Optimized native gel systems for separation of thylakoid ...

    Polyacrylamide gel electrophoresis (PAGE) is routinely used for protein analysis, and can also be used to separate nucleic acid fragments smaller than 100 bp. Nucleic acids are usually analyzed using a continuous buffer system where there is a constant buffer composition, pH, and pore size throughout the gel. Native proteins can be separated according to differences in their charge density, so long as the buffer in the gel is suitable for maintaining the protein in its native …

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  • Polyacrylamide Gel - an overview | ScienceDirect Topics

    2011-6-17 · Agarose gels can be used to resolve large fragments of DNA. Polyacrylamide gels are used to separate shorter nucleic acids, generally in the range of 1−1000 base pairs, based on the concentration used (Figure 1). These gels can be run with or without a denaturant. Gels that are run without a denaturant are referred to as native gels.

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  • Running agarose and polyacrylamide gels

    1984-1-1 · R.C. Allen, H.R. Maurer (Eds.), Electrophoresis and Isoelectric Focusing in Polyacrylamide Gel, de Gruyter, Berlin (1974)

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  • [12] Systems for polyacrylamide gel electrophoresis ...

    2013-9-24 · Laemmli’s system for polyacrylamide gel protein electrophoresis in the presence of the detergent SDS (SDS/PAGE) is one of the most cited methodological papers in life sciences . The facility with which SDS/PAGE resolves minute amounts of proteins revolutionized the analysis of tissue and cell extracts, resulting in “overnight” adoption of the technique in biochemistry, cell biology, immunology, …

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  • Acrylamide concentration determines the direction

    2006-6-27 · Remove water and fill the gaps to the left and right of the native gel strip using a 10% acrylamide native gel mixture (analogous to Step 1, Table 3). vii

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  • Blue native PAGE | Nature Protocols

    Add 400 µg mitochondria to 40 µL buffer A, 1 µg/mL leupeptin, 1 mM PMSF. Add 7.5 µL 10% LM and incubate on ice for 30 min. Centrifuge 72,000 x g at 4°C for 10 min. Add 2.5 µL of a 5% suspension of Coomassie blue G in buffer A. Load samples on 6 – 13% native acrylamide gradient gel.

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  • DNA Polyacrylamide Gel Electrophoresis

    2013-11-4 · DNA Polyacrylamide Gel Electrophoresis How to pour and run a neutral polyacrylamide gel. Buffers and Solutions Acrylamide:bisacrylamide (29:1) (30% w/v) Ammonium persulfate (10% w/v) Ammonium persulfate is used as a catalyst for the copolymerization of acrylamide and bisacrylamide gels. The polymerization reaction is driven by free

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  • SDS Polyacrylamide Gel Electrophoresis

    2011-7-14 · SDS Polyacrylamide Gel Electrophoresis Gel Recipes % Acrylamide 5% 7.5% 10.% 12.5% 15% 18% 4% Stacking Gel 30% Acrylamide (ml) 5.0 7.5 10.0 12.5 15.0 18.0 1.3 1% Bisacrylamide (ml) 7.8 5.8 3.9 3.1 3.1 3.1 1.5 1.5 M Tris, pH 8.7 (ml) 8.1 8.1 8.1 8.1 8.1 8.1 - 0.5M Tris, pH 6.7 (ml) - - - - - - 1.25 20% SDS (ml) 0.2 0.2 0.2 0.2 0.2 0.2 65 µl H 2

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  • A Guide to Polyacrylamide Gel Electrophoresis and

    2018-3-5 · Gel Electrophoresis: Separation of Native Basic Proteins by Cathodic, Discontinuous Polyacrylamide Gel Electrophoresis, bulletin 2376 10 11 Electrophoresis Guide Theory and Product Selection Two types of buffer systems can be used: Continuous buffer systems use the same buffer (at constant pH) in the gel, sample, and electrode

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  • Native PAGE Gels | Thermo Fisher Scientific - IN

    Add 7.5 µL 10% LM and incubate on ice for 30 min. Centrifuge 72,000 x g at 4°C for 10 min. Add 2.5 µL of a 5% suspension of Coomassie blue G in buffer A. Load samples on 6 – 13% native acrylamide gradient gel. Gel recipe and electrophoresis buffers described below.

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  • Blue native electrophoresis protocol | Abcam

    Peel the small piece of gel containing the DNA from the plastic wrap. 3. Transfer the gel slice to a microfuge tube. 4. Crush the gel slice against the wall of the microfuge tube with the disposable pipette tip. 5. Add 1 - 2 volumes of elution buffer to the gel slice (e.g., if the estimated gel slice volume is …

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  • Section VII: Separation of DNA in Polyacrylamide Gels

    2021-7-8 · Native polyacrylamide gel electrophoresis (native-PAGE) is an excellent tool for analyses of proteins and protein complexes in their native form. In early native-PAGE analyses, low concentrations of anionic detergents, such as sodium dodecyl sulphate (SDS), sodium deoxycholate (DOC) or

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  • Optimized native gel systems for separation of thylakoid ...

    2019-7-31 · In native PAGE electrophoresis most proteins have an acidic or slightly basic pl (isoelectric point) (~3–8) and migrate towards the negative polar. If your protein's pl is larger than 8,9, for example, you should probably reverse the anode and run the native PAGE gel. Learn more about Native-PAGE:

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  • Denaturing Polyacrylamide Gel Electrophoresis

    2019-2-5 · polyacrylamide around gel. 19. Hold two pieces of dry 46 × 57–cm blotting paper together as one piece. Beginning at one end of gel and working slowly towards the other, lay paper on top of gel. Take care to prevent air bubbles from forming between paper and gel. 20. Peel blotting paper up; gel should come off plate with it. Gradually curl ...

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  • NATIVE/MAIN GEL NATIVE/STACKING GEL

    2013-9-9 · NATIVE/MAIN GEL NATIVE/STACKING GEL dH 2O 4.11ml 6.2ml 1.5Tris pH:8.8 2..5ml 0.5 Tris pH: 6.8 2.5ml 30%Bis-Acrylamide 3.33ml 1.33ml 10%APS 50ul 50ul TEMED 5ul 5ul 5x Running buffer NATIVE(1lt) Tris 15g pH:8.3 Glycine 72g 5x Native Loading Dye(8.5): 1M Tris pH:6.8 2.5ml ...

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  • Blue native electrophoresis protocol | Abcam

    Add 7.5 µL 10% LM and incubate on ice for 30 min. Centrifuge 72,000 x g at 4°C for 10 min. Add 2.5 µL of a 5% suspension of Coomassie blue G in buffer A. Load samples on 6 – 13% native acrylamide gradient gel. Gel recipe and electrophoresis buffers described below.

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  • Gel Preparation for Native PAGE of DNA | National

    2012-7-23 · Gel Preparation for Native PAGE of DNA. Native PAGE gels are prepared by mixing an acrylamide/bisacrylamide monomer concentrate (AccuGel 19:1 or 29:1), buffer concentrate and water to achieve the desired gel concentration. TEMED and ammonium persulfate are added to initiate polymerization and the solution is poured into a cassette.

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  • Blue native PAGE | Nature Protocols

    2006-6-27 · Wittig, I. & Schägger, H. Advantages and limitations of clear native polyacrylamide gel electrophoresis. Proteomics 5 , 4338–4346 (2005). CAS PubMed Google Scholar

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  • Section VII: Separation of DNA in Polyacrylamide Gels

    Peel the small piece of gel containing the DNA from the plastic wrap. 3. Transfer the gel slice to a microfuge tube. 4. Crush the gel slice against the wall of the microfuge tube with the disposable pipette tip. 5. Add 1 - 2 volumes of elution buffer to the gel slice (e.g., if the estimated gel slice volume is …

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  • PAGE --- recipe calculator - Science Gateway

    Cell Biology Protocols. PAGE --- recipe calculator. Tris - glycine Native Polyacrylamide Gel (native-PAGE Calculator) Stacking. Separating. Gel percentage (%) Gel percentage (%) 30% Polyacrylamide (mL)

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  • BASIC PROTOCOL: PURIFICATION OF

    2016-8-9 · 3. Prepare the gelsolution (see Table 1 for appropriate acrylamide concentrations for resolvingsingle stranded DNAs). For a denaturing acrylamide gel of 20 cm x 16 cmx 1.6 mm, 60 ml of gel solution is sufficient, and it can be made by mixingthe following: 25.2 g urea (finalconcentration of 7 M) 6 ml 10x TBE buffer

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  • Protein Blotting Guide - Bio-Rad

    2017-1-4 · by native or SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and microfiltration is used to transfer proteins that are in solution. Detection The second phase, detection, entails probing the membrane with either a total protein stain or primary antibody specific to the protein of interest and subsequent visualization of the labeled proteins.

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  • Types of Western Blot Transfer Buffers | LSR | Bio-Rad

    Gels for isoelectric focusing, native PAGE, and those containing basic proteins or acid-urea may be transferred in 0.7% acetic acid. When using acetic acid for transfer, the proteins will be positively charged, so the membrane should be placed on the cathode side of the gel. General guidelines for transfer buffer and membrane selection by gel type.

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  • Native PAGE Separation Technique File No. 120

    Native PAGE Separation Technique File No. 120 PhastSystem™ 80-1311-96 Edition AB Gradient Gradient Gradient PhastGel Buffer Strips 4–15 10–15 8–25 SDS Native Gel description Dimensions (mm) 43 × 50 × 0.45 43 × 50 × 0.45 43 × 50 × 0.45 41 × 10 × 6 41 × 10 × 6 Gel material Acrylamide Acrylamide Acrylamide 3% Agarose 3% Agarose

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  • Blue native electrophoresis protocol | Abcam

    Add 7.5 µL 10% LM and incubate on ice for 30 min. Centrifuge 72,000 x g at 4°C for 10 min. Add 2.5 µL of a 5% suspension of Coomassie blue G in buffer A. Load samples on 6 – 13% native acrylamide gradient gel. Gel recipe and electrophoresis buffers described below.

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  • Polyacrylamide Gel Electrophoresis (PAGE)

    2006-12-13 · POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) Last Update: December 2006. PREPARE SOLUTIONS. 1. 30% Acrylamide 0.8% Bis (300 mL): Mix 90 g of Acrylamide, 2.4 g of N'N'-bis-methylene-acrylamide, and 300 mL of dH 2 O (filter through a 0.45 m filter) (Store at 4 o C in a dark bottle) 2. 10% SDS (100 mL): Weigh 10 g of SDS and add dH 2 O to 100 mL.

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  • Optimized native gel systems for separation of thylakoid ...

    2021-7-8 · Native polyacrylamide gel electrophoresis (native-PAGE) is an excellent tool for analyses of proteins and protein complexes in their native form. In early native-PAGE analyses, low concentrations of anionic detergents, such as sodium dodecyl sulphate (SDS), sodium deoxycholate (DOC) or

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  • Blue native electrophoresis protocol - Abcam

    2015-6-12 · Native acrylamide gels can be poured by hand. While it is possible to use a single acrylamide concentration such as a straight 10% gel, we highly recommend the use of a linear acrylamide concentration such as 6-13%. A recipe for pouring these native acrylamide gels in a 10-gelBioRad Mini-PROTEAN II multicasting chamber when

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  • A rapid method for isoelectric focusing in

    1972-7-15 · The gel slab will measure about 190 X 95 X 1.5 mm. The following recipe was North-Holland Publishing Company - Amsterdam used to prepare one gel plate: 1.4 ml Ampholine (Amph) 3-10, 0.10 ml Amph 4-6, 0.10 ml Amph 5-7, 0.20 ml Amph 8-10, 0.4 g glycine, 0.40 ml riboflavin (from a stock solution of 4 mg 100 ml) and 25 ml distilled water.

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  • Denaturing Polyacrylamide/Urea Gel Electrophoresis

    2018-8-22 · Heat the gel (buffer) during the whole run at 60-70°C. 9. Wash the wells with 1X TBE buffer to remove UREA and gel pieces. 10. Load the samples. 11. Run the gel at 6 V/cm till the lower dye front reaches the three thirds of the gel. 12. Soak the gel for about 15 min in …

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  • Denaturing Urea PAGE - Small Gel

    2001-6-22 · Denaturing Urea PAGE - Small Gel 1. Prepare denaturing polyacrylamide gel solution. Use Gibco/BRL apparatus. 7.2 % 9.6 % 12 % 10X TBE 2.5 mls 2.5 mls 2.5 mls Urea (ultrapure) 10.5 g 10.5 g 10.5 g 40% Acrylamide 4.5 mls 6 mls 7.5 mls ddH2O 10.5 mls 9 mls 7.5 mls Total Volume 25 mls 25 mls 25 mls Use 40% acrylamide stock for DNA/RNA gels.

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  • Native SDS-PAGE: high resolution electrophoretic ...

    2014-3-13 · Introduction. The most commonly used technology to obtain high resolution analytical separation of mixtures of proteins is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 1, 2 The procedure involves initial denaturation of component proteins with an anionic detergent that also binds to them, imparting to all proteins a negative charge proportional to their molecular mass.

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  • Protein gel electrophoresis technical handbook

    2021-7-12 · Polyacrylamide gel electrophoresis (PAGE) ... an anionic detergent such as sodium dodecylsulfate (SDS). SDS denatures and unfolds the protein by wrapping around the hydrophobic portions. SDS binds at a ratio of ~1.4 g SDS per ... The combination of a lower-pH gel buffer (pH 6.4) and running buffer (pH 7.3–7.7) leads to a significantly lower ...

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  • NativePAGE Novex Bis-Tris Gel System

    2012-3-27 · polyacrylamide mini gel system to perform native (non-denaturing) electrophoresis. The near neutral pH 7.5 environment during electrophoresis results in maximum stability of both proteins and gel matrix, providing better band resolution than other gel systems including the traditional Tris-glycine native electrophoresis (Laemmle) system.

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  • Denaturing Urea PAGE - Small Gel

    2001-6-22 · Denaturing Urea PAGE - Small Gel 1. Prepare denaturing polyacrylamide gel solution. Use Gibco/BRL apparatus. 7.2 % 9.6 % 12 % 10X TBE 2.5 mls 2.5 mls 2.5 mls Urea (ultrapure) 10.5 g 10.5 g 10.5 g 40% Acrylamide 4.5 mls 6 mls 7.5 mls ddH2O 10.5 mls 9 mls 7.5 mls Total Volume 25 mls 25 mls 25 mls Use 40% acrylamide stock for DNA/RNA gels.

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  • (PDF) A Guide to Polyacrylamide Gel Electrophoresis

    For protein separation, virtually In gel electrophoresis, proteins do not all enter the all methods use polyacrylamide as an anticonvective, gel matrix at the same time. Samples are loaded sieving matrix covering a protein size range of into wells, and the proteins that are closer to the gel 5–250 kD.

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  • Tris–Acetate Polyacrylamide Gradient Gels for the ...

    2012-4-9 · Abstract. Polyacrylamide gel electrophoresis (PAGE) is one of the most powerful tools used for protein analysis. We describe the use of Tris–acetate buffer and 3–15% polyacrylamide gradient gels to simultaneously separate proteins in the mass range of 10–500 kDa.

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  • Polyacrylamide Gel Electrophoresis (PAGE ...

    SDS-PAGE Gel. 1. Prepare the separation gel (10%). Mix in the following order: After adding TEMED and APS to the SDS-PAGE separation gel solution, the gel will polymerize quickly, so add these two reagents when ready to pour. 2. Pour gel, leaving ∼2 cm below the bottom of the comb for the stacking gel. Make sure to remove bubbles.

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  • SDS-PAGE Gel - CSH Protocols

    2013-9-24 · Gel Mobility of TM-Mimetics Relative to Reference Proteins Changes with Acrylamide Concentration. The set of TM protein mimetics we designed and prepared are polymers of a peptide with the core sequence NH 2-SKSKS-Leu 20-SKSKS-NH 2, termed “pL 20 ” ().The average length, high hydrophobicity, and abundance of Leu in natural membrane-spanning regions are recapitulated in the …

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  • Acrylamide concentration determines the direction

    2016-7-13 · How SDS-PAGE Works. SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) is commonly used in the lab for the separation of proteins based on their molecular weight. It’s one of those techniques that is commonly used but not frequently fully understood. So let’s try and fix that. SDS-PAGE separates proteins according to ...

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  • Coomassie Blue (R-250, G-250)

    2020-11-2 · 3- Destain gel in 10% acetic acid for 2 hours or more. Store gels in 7% HOAC. Blue Native PAGE Taking to good account the negative charge of Coomassie dye bound to proteins, the Coomassie can be used to separate protein complexes using polyacrylamide gel electrophoresis under non-denaturing conditions in a technique called Blue Native PAGE.

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  • Native SDS-PAGE: high resolution electrophoretic ...

    2014-3-13 · Introduction. The most commonly used technology to obtain high resolution analytical separation of mixtures of proteins is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 1, 2 The procedure involves initial denaturation of component proteins with an anionic detergent that also binds to them, imparting to all proteins a negative charge proportional to their molecular mass.

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  • Chapter 2 : Protein Electrophoresis

    2009-12-14 · separated on this type of gel. *At the usual pH values between 6.8 and 8.8, Tricine migrates much faster than glycine in a stacking gel despite its higher molecular mass, because much more Tricine was in a the migrating, anionic form.

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  • Nioplexes encapsulated in supramolecular hybrid ...

    The resultant complexes were analyzed by using electrophoresis on a 20% polyacrylamide gel at 150 V for 6 h at 25 °C in 1× TBE buffer. The polyacrylamide gel electrophoresis (PAGE) was immersed in 1× TBE buffer (200 μL) and stained with SYBR-Green (20 μL) (shaking 15 min at room temperature).

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  • Detergents - ITW Reagents

    Used in polyacrylamide gel electrophoresis for proteins, which show an unusual migration behavior in the SDS-PAGE (e. g. strongly charged proteins or subunits of membrane proteins). Another important application of CTAB is the precipitation of high molecular weight DNA, especially from plant material.

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  • Western blot sample preparation | Abcam

    To denature, use a loading buffer with the anionic detergent sodium dodecyl sulfate (SDS), and boil the mixture at 95–100°C for 5 min. Heating at 70°C for 5–10 min is also acceptable and may be preferable when studying multi-pass membrane proteins. These tend to aggregate when boiled and the aggregates may not enter the gel efficiently.

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  • 'gel' Protocols and Video Articles

    DNA Isolation Expand NOTE: In this experiment you will perform DNA isolation under two experimental conditions: one using a buffer containing the detergent SDS and one without det

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  • Buffer system for electrophoresis and use thereof ...

    1997-5-30 · Polyacrylamide gel is frequently used due to its high chemical and mechanical stability. Polyacrylamide gel electrophoresis is often referred to as PAGE. Proteins and peptides are usually denatured and treated with sodium dodecyl sulphate (SDS), an anionic detergent, prior to the electrophoresis, so-called SDS electrophoresis.

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  • 'protein complexes' Protocols and Video Articles

    Video articles in JoVE about 'protein complexes' include '2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes', 'Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling', 'Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of ...

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  • Special Interaction of Anionic Phosphatidic Acid

    2014-7-15 · Anionic phosphatidic acid (PA) has been shown to stabilize and bind stronger than phosphatidylglycerol via electrostatic and hydrogen bond interaction with the positively charged residues of potassium channel KcsA. However, the effects of these lipids on KcsA folding or secondary structure are not clear. In this study, the secondary structure analyses of KcsA potassium channel was carried …

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  • Cell Lysis Solutions | Thermo Fisher Scientific - US

    2014-3-13 · Introduction. The most commonly used technology to obtain high resolution analytical separation of mixtures of proteins is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 1, 2 The procedure involves initial denaturation of component proteins with an anionic detergent that also binds to them, imparting to all proteins a negative charge proportional to their molecular mass.

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  • Native SDS-PAGE: high resolution electrophoretic ...

    Used in polyacrylamide gel electrophoresis for proteins, which show an unusual migration behavior in the SDS-PAGE (e. g. strongly charged proteins or subunits of membrane proteins). Another important application of CTAB is the precipitation of high molecular weight DNA, especially from plant material.

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  • Detergents - ITW Reagents

    Example gel stained with a zinc stain. A 2-fold dilution series of a protein mixture was separated by protein gel electrophoresis using a 15-well mini gel. Subsequently the gel was stained using the Thermo Scientific Pierce Zinc Reversible Stain Kit, and then photographed with the gel placed over a dark blue background. The sensitivity on this ...

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  • Protein Gel Staining Methods | Thermo Fisher

    1999-1-11 · The electrophoresis gel may be any agarose or polyacrylamide gel. Preferably, the electrophoresis gel comprises between 3% and 25% (%T) acrylamide polymerized using from about 1% to about 6% cross linker (%C). More preferably, this polyacrylamide gel is polymerized using from about 2% to about 5% crosslinker (%C).

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  • 9 Transfer the gel to the histochemical stain Watch for ...

    2014-1-13 · Repeatsteps 4-6 acetonewashes. Drypellet placingtube 95Cheat block 5-10min driveoff acetone. SDS-PAGE,add 2X 4Xsample buffer withoutbME) boilsmaple 10min 95Cherat block before loading smaple onto polyacrylamide gel.

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  • System for pH-neutral stable electrophoresis gel -

    Using a clean razor blade, cut the protein bands of interest from the gel, eliminating as much polyacrylamide as possible. Place the gel slices in a 0.5ml microcentrifuge tube prewashed twice with 50% ACN/0.1% TFA. Destain the gel slices with 0.2ml of 100mM NH 4 HCO3/50% ACN for 45 minutes at 37°C. Repeat this destaining step once.

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  • Protease Digestion for Mass Spectrometry | Protein

    For digestion of native proteins, dissolve the protein in 50mM NH 4 HCO 3 (pH 7.8) or 50mM Tris-HCl (pH 8) without urea. Add Trypsin Gold, Mass Spectrometry Grade, to a final protease:protein ratio of 1:100 to 1:20 (w/w). Incubate overnight at 37°C.

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  • WO2007121981A1 - Cationic polysaccharide, its

    The present invention relates to polysaccharide, preferably starch, of high purity having cationic degree of substitution over 0.50 and bound nitrogen index after cationisation reaction stage over 0.75, and to preparation thereof as well as to its use in making of paper or paperboard.

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