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very convenient tool for evaluating sugar chain - protein interaction

Gold Nano Particles for Bioseparation SGNP (Sugar chain immobilized Gold Nano Particle)


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Background

SGNP (Sugar chain - immobilized Gold Nano-Particle) is a gold nano particle immobilized with structurally defined sugar chains, showing purple-red color (Abs. max = ca. 530 nm).@In the present product (Heparin-GNP), heparin, a sulfated polysaccharide, is immobilized.The diameter of particle is ca.15 to 30 nm.

Application

1. Aggregation assay of protein(s)
2. Inhibition assay
3. Probe for Dot-Blotting assay
4. Isolation and identification of target protein from a crude extract

[Application#1: Aggregation assay of protein(s)]
Regularly sugar chain binding proteins, such as lectins, possess multiple binding sites for sugar chains. By the addition of SGNP to the protein solution, the protein may form aggregates with sugar chains immobilized SGNP in a short time of period. The change can be seen visually, or can be quantified by measuring OD at around 530 nm. Using SGNP, the binding properties (selectivity, dissociation constant (KD), specificity, etc.) are easily evaluated.

<Recommended protocol>

Dissolved SGNP at Abs530nm  3.0 using your buffer. *see Amount of SGNP

1. In wells of 96-well microtiter plate (round bottom), 25µl of protein solution was added with changing the concentration. *Sequential 1:2 dilution from ca. 200 µg/ml (or ca. 4 µM), 6 points or more.
2. Addition of 25 µl of SGNP solution prepared as above. Gentle agitation for 0.5 to 2 hr at room temperature.
3. Measure OD at 530 nm of the supernatant.

<Example>
  Final concentration of Sample (nM) OD530 nm Measument
2,000 ---------> 32.3
Alpha-Glucosyl-GNP
(Cat. No. G-00M-100)
Con A
RCA 120
BSA
Beta-Galactosyl GNP
(Cat. No. G-00L-100)
Con A
RCA 120
BSA

* Con A<Concanavalin A> (EY LABORATORIES, Cat. No.L1104250) RCA120<Ricinus communis Agglutinin I > (Vector, Cat. No.L-1080) BSA <Albumin, bovine serum> (SIGMA Aldrich, Cat. No.A0281)

[Application#2: Inhibition assay]
This assay is useful to know the specificity of the ligands for the target protein. By the addition of inhibitor (mono-saccharide, oligo-saccharide, mimetic compounds, glycoprotein, or drug candidate), the formed protein-SGNP aggregates may be re-dissolved by the competitive binding of protein with the inhibitor.


1. Dissolve SGNP at Abs530nm  4.0 ` 6.0 using your buffer *see Amount of SGNP
2. In wells of 96-well microtiter plate (round bottom), 25µl of protein solution (Conc. > KD) and 25 µl of SGNP solution as prepared above were added. *It is important to know KD value of the protein against sugar chain on SGNP
3. 50 µl of the inhibitor dissolved in the same buffer (conc. 0.1 ` 50 mM) is added to the above mixture. Then, agitate for 1 hr or overnight.
4. Measure OD at 530 nm of the supernatant.

[Application#3: Probe for Dot-Blotting assay]
The sugar chain binding potency of the trace amount of your samples can be detected. * Independent on the valency of sugar-binding proteins

<Recommended protocol>

1. Prepare 10`20ml SGNP solution of Abs530nm  0.15 ` 0.30 using your buffer.
*see Amount of SGNP
2. On nitrocellulose membrane* (10 x 30 mm), spot your sample (0.3 – 1 mL, 0.1- 2.0 mg) and dry up at room temperature. 
*Trans-Blot™ Transfer Medium,@Pure Nitrocellose Membrane (0.2 mm) (Bio-Rad, Cat. No.162-0146)
3. To 10 mL of SGNP solution in a glassware (φ 50 mm), the membrane is soaked with a gentle agitation for 5 – 30 min.
*You may need to check the staining every 5 min.  Please anchor the membrane with tweezers
4. Wash the membrane with buffer and dry up.
*If you see too high background, decrease the concentration of SGNP or shorten the soaking time.

<Example>
Sample
SGNP
Con A WGA RCA 120
Alpha-Glucosyl-GNP
(Cat. No. G-00M-100)

Beta-Galactosyl-GNP
(Cat. No. G-00L-100)

Beta-GlcNAc-GNP
(Cat. No. G-AGN-100)

* Con A<Concanavalin A> (EY LABORATORIES, Cat. No.L1104250) RCA120 <Ricinus communis Agglutinin I > (Vector, Cat. No.L-1080) WGA (Wheat Germ) (Seikagaku Kogyo, Cat. No.5400)

[Application#4: Isolation and identification of target protein from a crude extract]
Quick isolation and identification of protein(s) having multi-binding sites (lectin) from a plant-derived crude extract of cell-lysate are available using SGNP. Once you get an aggregate by mixing appropriate SGNP with your extract or lysate, the aggregate can be directly applied for SDS-PAGE. Then, the protein band(s) in SDS-gel is analyzed according to the proteomics procedure

<Recommended protocol>
Quick isolation and identification of protein(s) having multi-binding sites (lectin) from a plant-derived crude extract of cell-lysate are available using SGNP. Once you get an aggregate by mixing appropriate SGNP with your extract or lysate, the aggregate can be directly applied for SDS-PAGE. Then, the protein band(s) in SDS-gel is analyzed according to the proteomics procedure.

1. Dissolve SGNP at Abs530nm  3.0 using your buffer
*see Amount of SGNP
2. To 50 ml of SGNP solution in a 1.5 mL of eppendorf tube, add 50 ml of your extract.  The mixture is incubated for 1 h to overnight at 4 degree C with a gentle agitation.
* The protein concentration in the extract may be in the range between 500 and 10 mg/mL.
3. Centrifugation at 6,000 ` 10,000 x g for 10 min, and remove the supernatant.
4. To the precipitate, add 500 ml of buffer, vortex for 10 sec and centrifuge at 6,000 ` 10,000 x g for 10 min, and remove the supernatant.  This may repeat 2 more times.
5. To the precipitate, add 10-30 ml of sample preparation buffer*, boiled up for 10 min..
*Laemmli Sample Buffer (Bio-Rad, Cat. No.161-0737)
6. SDS-PAGE
*Reducing or non-reducing condition can be used.

<Example>

Product List

Product Name Cat# Quantity Price

(α)Glucosyl GNP ,

SXB-G-00M-250-EX

1 VIAL ¥7,000
$94

(β)Glucosyl GNP ,

SXB-G-00C-250-EX

1 VIAL ¥7,000
$94

(α)Galactosyl GNP ,

SXB-G-00E-250-EX

1 VIAL ¥7,000
$94

(β)Galactosyl GNP ,

SXB-G-00L-250-EX

1 VIAL ¥7,000
$94

(α)GlcNAc GNP ,

SXB-G-AGN-250-EX

1 VIAL ¥18,000
$240

(β)GlcNAc GNP ,

SXB-G-BGN-250-EX

1 VIAL ¥18,000
$240

(α)GalNAc GNP ,

SXB-G-AAN-250-EX

1 VIAL ¥18,000
$240

(β)GalNAc GNP ,

SXB-G-BAN-250-EX

1 VIAL ¥18,000
$240

(α)Fucosyl GNP ,

SXB-G-0AF-250-EX

1 VIAL ¥18,000
$240

(β)Fucosyl GNP ,

SXB-G-0BF-250-EX

1 VIAL ¥18,000
$240

(α)Mannosyl GNP ,

SXB-G-AMA-250-EX

1 VIAL ¥9,000
$120

3SialylGalactosyl GNP ,

SXB-G-03S-250-EX

1 VIAL ¥18,000
$240

6SialylGalactosyl GNP ,

SXB-G-06S-250-EX

1 VIAL ¥18,000
$240

SGNP set : *Each vial in a set contains SGNP showing Abs530nm = 3.0m/cmn, when the whole SGNP is dissolved in 0.25 mL of buffer


Product Name Cat# Quantity Price

SGNPs#1 [Sugar immoblized Gold Nano particles #1] DataSheet ,

SXB-G-AB1-250-EX

1 SET ¥140,000
$1867

SGNPs#2 [Sugar immoblized Gold Nano particles #2] DataSheet ,

SXB-G-AB2-250-EX

1 SET ¥170,000
$2267

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.



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