Vector Laboratories, Inc. - PowerPoint Presentation

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Vector Laboratories, Inc.

State of the Art Labeling and Detection Systems

Burlingame, California, U.S.A.

www.vectorlabs.com

Established 1976, California, U.S.A.

Privately-held

Recently Acquired by Maravai Life Sciences in collaboration with GTCR, a private

e

quity companyManufacturer and retailer of approx. 600 products for biological and medical research applications

Two subsidiaries: Vector Canada and Vector U.K.

Over 50 worldwide distributors

Pioneered biotin/avidin detection methods and patented Vectastain® ABC system

ABC-Amp

Animal-Free Blocker

AT-Yellow

BLOXALL

DuoLuXEliteEndTag

FastTag

Fusion Aid

GC-RedHYBEXImmEdgeImmPACTImmPRESS

ImmPrintM.O.M.NeurobiotinNicKit p.s.o.

Photoprobe

PolyBlock

ProFusion

ProtOnQuant*TagResolve-ItUltraSNAPVectabondVectaMountVectashieldVectashield Hard*SetVectastainVectorVector BlackVector BlueVector NovaREDVector RedVectrex

Immunohistochemistry / Immunocytochemistry

Immunofluorescence

In Situ

Hybridization

Neural TracingDNA/RNA Labeling

Western / Southern Blotting

ELISAs

Flow CytometryLectin and glycoprotein binding

Lectin Overview

The term “lectins” are today regarded as essentially proteins that bind to carbohydrates (sugars).

Lectins are found in plant, animal and other sources.

Vector currently offers about 40 plant only derived lectins.

All Vector lectins are extracted from the native plant source (i.e. seeds, bulbs, fruit etc.) and are not recombinant.

The biological significance of lectins in plants remains unclear, however isolated lectins show mitogenic or agglutination activity or even toxic effects on cells.

The sugar binding properties of lectins make them a useful tool for identifying or isolating defined glycoproteins.

Unconjugated Lectins

Applications

:

Largely for cell based assays (e.g. growth/inhibition actions).

Custom conjugation applications by end-user.

Plated (e.g. microarray) for capture/screening of glycoproteins.

I

njected into live animal models and subsequently detected using specific anti-lectin antibody.

Example

: PHA-L (L-1110), ionotopherically injected

into single neuron for transport (survival

time) then visualized using anti-PHA antibody (e.g. BA-0224) in excised, fixed tissue.

Fluorophore conjugated Lectins

Single step application/visualization.

Expanding range of fluorophore choices:

Traditional: Fluorescein, rhodamine

Contemporary: DyLight Fluors (488, 594)

In vitro

studies include frozen/paraffin sections, flow cytometry

In vivo

perfusion studies

(e.g. angiogenesis in tumor/xenograft rodent models). Left image: Mouse retina perfused with DyLight 594-LEL (DL-1177, red), mounted with Vectashield HardSet with DAPI (H-1500, blue). Right image: Mouse kidney (paraffin section) stained with DyLight 488-LEL (DL-1174, green), mounted with Vectashield HardSet with DAPI

(H-1500

, blue).

Biotinylated Lectins

Offer the most flexibility for subsequent visualization or detection using (strept)

avidin

conjugates, since limited anti-lectin antibodies.

Examples: Streptavidin peroxidase (HRP) + DAB substrate (IHC or Blotting)

Avidin Fluorescein (IF visualization)

Streptavidin coated magnetic beads (Capture) Two-step method provides greater sensitivity over directly conjugated format.

Left

image

:

Colon section stained with DBAlectin (black, DAB Ni++) and Alcian Bluecounterstain.Right image:Sheep trachea stained with PNA lectin (brown, DAB).

Lectin tissue staining guidelines

Avoid the use of sera

(i.e. normal animal serum)

, milk derived reagents

(non-fat dry milk or casein), or any solution containing potential sugars or glycoproteins as blocking agents or included in buffer diluent.

Use high grade BSA

(SP-5050)

as blocking/diluent alternative or Carbo-Free blocker (SP-5040).

High temperature antigen unmasking (paraffin sections) not required.

Controls: Positive

(known tissues from published literature)

Negative

(pre-adsorb working dilution with defined sugar).Room Temp., 30-60 min incubation (standard slide mounted section).Working dilution ~ 5 to 20 µg/ml conc. in PBS / TBS buffer

Agarose bound Lectins

Supplied as a hydrated matrix

1:1 – settled agarose lectin : buffer

Usually applied to a column format

(see image)

or in a dish as a slurry.

Allows for capture/isolation of glycoproteins that, once bound to lectin, can be eluted off

(i.e. reversible binding)

using defined high concentration sugar solutions

(e.g. glycoprotein eluting solutions such as ES-1100)

.

Can be re-used for same prep multiple times.Can be stored in fridge for extended periods.Column

Agarose

Lectin

Collecting

Tubes

Glycobiology : Eluting Solutions

Glycoprotein Eluting Solutions:

Catalog Number

ES-1100 for use with Mannose- and Glucose-binding lectins

ES-2100 for use with Galactose- or GalNAc-binding lectins ES-3100 for use with Fucose- and Arabinose-binding lectins ES-5100 for use with GlcNAc- or Chitin-binding lectins

ES-7100 for use with Sialic Acid-binding lectinsConsist of concentrated defined sugars and salts

Proprietary make-up / recipe for each solution

Supplied as 100 ml ready-to-use solutions

Single

use solutions (i.e. do not reuse collected solution once exposed to column)May require several aliquot “passes” over column for maximum recovery of bound glycoprotein Eluting solutions also “regeneration” solutions and retain integrity of column without loss of binding capacity

Glycobiology : Neoglycoproteins

Neoglycoproteins

Catalog

Number

BSA : Sugar Conjugate

G-1000

BSA-Mannose

G-2000

BSA-Galactose

G-3000

BSA-

Fucose

G-4000BSA-GalNAcG-5000BSA-ChitinG-6000BSA-GlcNAc

G-7000

BSA-Sialic Acid

G-8000

BSA-Arabinose

G-9000

BSA-Glucose

Applications

Positive

controls for agarose lectin columns

Positive

controls for lectin blot applications

Localize endogenous “lectins” or sugar binding molecules in cell or tissue preps.

Can be conjugated for subsequent detection/visualization

Approx. 22 : 1 ratio (sugar to BSA)

Catalog Number

Sugar

S-9001

N-

acetylgalactosamine

S-9002

N-acetylglucosamine

S-9003GalactoseS-9004

Lactose

S-9005

α

- methylmannosideS-9006α - methylglucosideS-9007L-fucoseS-9008N-acetylneuraminic acid

(sialic acid)

Glycobiology : Inhibiting Sugars

Application

For use as

negative

controls:

Dilute lectin conjugate to working concentration with buffer containing approx. 200 mM of sugar specific for the given lectins binding affinity (i.e. AAL use product S-9007 as AAL is a

fucose

binding lectin).

Incubate this lectin : sugar solution on the bench (R/Temp) for ~30-60 min to “absorb”.

Apply “bound” lectin to cell/tissue prep. Should observe significant drop in binding

compared with non-absorbed lectin.

Inhibiting Sugars

Glycobiology : Agarose bound Sugars

Catalog Number

Agarose Sugar

AC-1001

Galactose Agarose

AC-1002

Fucose Agarose

AC-1003

Lactose Agarose

AC-1004

Mannose Agarose

AC-1005

N-acetylgalactosamine AgaroseAC-1006N-acetylglucosamine AgaroseAC-1007Raffinose AgaroseAC-1008Sialic Acid Agarose

Agarose bound Sugars

Applications

Capture/isolate sugar binding proteins (i.e. lectins)

*

Positive

control to determine lectin binding

Usually applied in a column

Bound material can be eluted via high sugar concentration

Can re-use for same prep.

*

This application is a “reverse” of the agarose bound lectins with

the understanding that given lectins may bind more complex structures

than the simple sugars bound to agarose.

Glycobiology : Lectin Screening Kits

Lectin Screening Kits

I

*

Lectin Screening Kits II

#

Lectin Screening Kits III

¥

Unconjugated Lectins

LK-2000

LK-3000

Biotinylated Lectins

BK-1000

BK-2000

BK-3000

Fluorescein Lectins

FLK-2100

FLK-3100

FLK-4100

Rhodamine Lectins

RLK-2200

RLK-3200

Lectin Format

*

Lectin Screening Kits I

: consist of 1 mg each of Con A, DBA, PNA, RCA I, SBA, UEA I and WGA lectins or lectin conjugates

#

Lectin Screening Kits II

: consist of 1 mg each of GSL I, LCA, PHA-E, PHA-L, PSA,

Succ

. WGA and SJA lectins or lectin conjugates

(

note SJA not included in FLK-3100)

¥

Lectin Screening Kits III

: consist of 0.5 mg each of DSL, ECL, GSL II,

Jacalin

, LEL, STL and VVL l lectin conjugates

Glycobiology : Antibodies to Lectins

Offer 12 unconjugated goat polyclonal (affinity purified ) anti-lectin antibodies. One of these is also made in rabbit.

Of these 12, five are also offered as biotinylated formats

The most commonly used are anti-PHA (E+L)

and anti-WGA for the detection of neuron

injected / transported unconjugated lectin

Note that WGA is the only lectin to offered as an HRP conjugate

f

or direct injection / detection (ready-to-use, PL-1026)

Other

applications would include detection of bound lectin such as in a microarray or ELISA platform.

Additional Resources

Vector Website

(

Support Menu):

- Protocols: Recommended

procedures

(http://vectorlabs.com/protocols)

- Brochures: Lectins and Glycobiology (pdf for download)

(

http

://

vectorlabs.com/brochures) - Individual lectin descriptions and data sheets (sugar specificity, format, volume, etc.) Published References: - Literature Searches (e.g. Google Scholar for defined applications)Technical Service