Sharp R-1501C User Manual download pdf

EY LABORATORIES, INC.

107 North Amphlett Blvd.

San Mateo, CA 94401

Tel:

Fax:

Orders:

650-342-3296

650-342-2648

1-800-821-0044

(Outside CA only)

EY LABORATORIES, INC.

107 North Amphlett Blvd.

San Mateo, CA 94401

Tel:

Fax:

Orders:

650-342-3296

650-342-2648

1-800-821-0044

(Outside CA only)

PRODUCT INFORMATION

TRITC Labeled Lectin

Catalog Number: R-1501-1

Description: Pure Limulus polyphemus lectin (LPA) from horseshoe crab, TRITC conjugated.

Lot Number:

Protein Concentration:

(Based on OD 280)

1 mg purified LPA TRITC / 1 ml Buffer.

TRITC / Protein Ratio:

(OD 550 / OD 280)

Purification Procedure: Gel filtration performed after conjugation to remove free TRITC.

Carbohydrate

Specificity:

Sialic Acid (N-Acetyl neuraminic acid).

Inhibitory

Carbohydrate:

N-Acetylneuraminic acid and N-Glycolylneuraminic acid.

Activity: 10-20 g/ml will agglutinate type O human erythrocytes. As much as 100 g/ml

may be necessary to agglutinate type A or B cells.

Buffer:

0.05M Tris - 0.15M NaCl, 0.01M CaCl

, pH 8.0. Contains 0.05%sodium azide as a

preservative.

Chemical Used for

Conjugation:

Tetramethylrhodamine Isothiocyanate, TRITC.

Storage: Store liquid material refrigerated in aliquots in amber vials or covered with foil.

DO NOT FREEZE.

Stability: The liquid material is stable for at least 1 year when stored refrigerated in aliquots

with 0.05% sodium azide added as a preservative.

Caution: Refer to the enclosed MSDS for information regarding Lectins. The aluminum

seals have sharp edges and the vial itself may have cracks which can cause

lacerations. Use caution when opening the vial.

Remarks: Calcium is REQUIRED for binding. The addition of millimolar concentrations of

sialic acid in the above buffer of the addition of a calcium chelting agent such as

EDTA may be used to inhibit binding. LPA is composed if 18-20 noncovalently

bound subunits and may precipitate if frozen. Clarify by low speed centrifugation.

Fluorescent Conjugates are extremely light sensitive.

References: 1. Roche, A. C., et.al. (1974) Biochem. Biophys. Acta. 371:242-254.

2. Marchalonis, et.al. (1968) J.Mol.Biol.32:453.

3. Roche, A., et.al. (1975) FEBS Lett. 57:245.

4. Lotan, R., et al. (1977) Biochem. 16, 9:1787.

5. Robey, F.A. et.al. (1981) J. Biol. Chem. 256:969-975.

6. Pardoe, G.I., et.al. (1970) Immunol.18:73-83.

General Procedure

Fluorescent Labeled Lectin

The following is a general Procedure and Trouble-Shooting Guide. The information is provided only for

your convenience. The success of your experiments are not guaranteed by EY Laboratories, Inc.

Tissue Sections

1. Wash and block tissue section. Do not use serum products, they contain glycoproteins which may lead

to high levels of non specific background. After blocking, rinse briefly with Buffer (See reverse side).

2. Dilute Fluorescent Labeled Lectin to desired concentration 20-100 g/ml using Buffer.

3. Incubate tissue section with Fluorescent Labeled Lectin for 30 minutes in a moist chamber.

4. Wash tissue section with Buffer three times.

5. Examine tissue section with Fluorescent microscope. Use appropriate filter.

Ref. M. Immbar et. al., (1973). Intnl. Journal of Cancer, 12, 93-99

Cell Suspension

1. Wash cells with Buffer (See reverse side.)

2. Collect cells by centrifugation.

3. Dilute Fluorescent Labeled Lectin to 100



g/ml using Buffer.

4. Incubate approximately 1x10

cells with 1 ml diluted Fluorescent labeled Lectin for 15 minutes at room

temperature or in a 37°C water bath.

5. Wash cells with Buffer three times using centrifugation.

6. Examine cells, with or without fixation with Fluorescent microscope. Use appropriate filter.

Ref. K. Phiss. (1977). Experimental Pathology, 14, S15

Fluorochromes must be protected from light. Perform incubation, when practical, in a dark room or

covered in foil.

Absorption and Emission

Absorption/Excitation Rate Emission Max.

FITC 492 nm 517 nm

TRITC 554 nm 570 nm

Texas Red™ 596 nm 615 nm

Carbohydrate Inhibition

Inhibition of lectin binding may be accomplished by using one of two procedures:

A. Before incubating with Fluorescent Labeled Lectin, incubate section or cells with inhibitory

carbohydrate for 30-60 minutes at room temperature. NOTE: Complete inhibition may NOT occur.

B. Preincubate diluted Fluorescent Labeled Lectin with inhibitory carbohydrate for 30-60 minutes at

room temperature before applying to section or cells.

TROUBLE SHOOTING GUIDE

Problem Cause Solution

1. Low concentration of specific

oligosaccharide on sample.

Causes #1 - #3

a. Increase incubation time.

2. Low concentration of lectin conjugate. b. Increase concentration conjugate.

Weak or no

Staining

3. Insufficient incubation time.

4. Photobleaching a. Avoid exposure to light.

1. Lectin conjugate is too concentrated. a. Decrease concentration of Lectin conjugate.

b. Shorten incubation times.

2. Insufficient washing. a. Perform multiple washings and prolong

High washing time.

Background 3. Autofluorescent sample. a. Use fluorochrome with different excitation

and emission spectrum.

b. Use a different lectin conjugate (enzyme or

colloidal gold).

a. Perform control reactions.

Multiple causes b. Use other cytochemical technique to prove

Unexpected

Staining Pattern

or disprove the findings.

Sample Only

1 2

Sample Only 1

Summary of Contents

Page 1 - Sample Only

EY LABORATORIES, INC.107 North Amphlett Blvd.San Mateo, CA 94401Tel:Fax:Orders:650-342-3296650-342-26481-800-821-0044(Outside CA only)EY LABORATORIES,

Page 2

EY LABORATORIES, INC.107 North Amphlett Blvd.San Mateo, CA 94401Tel:Fax:Orders:650-342-3296650-342-26481-800-821-0044(Outside CA only)EY LABORATORIES,

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Sharp R-1501C User Manual

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