Galectin 3 Protein (LGALS3)

Produktnummer ABIN5509728

Produktdetails

Target: Galectin 3 (LGALS3)

Protein-Typ: Recombinant

Biologische Aktivität: Active

Spezies: Human

Quelle: Escherichia coli (E. coli)

Applikation: Functional Studies (Func)

Produktmerkmale: Recombinant Human Galectin-3 (LGALS3)

Reinheit: >95 % as determined by SDS-PAGE

Endotoxin-Niveau: < 1.0 EU of endotoxin/μg of protein

Anwendungsinformationen

Applikationshinweise: Optimal working dilution should be determined by the investigator.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (H)

Validierung #103788 (Haemagglutination)

by: Sanja Dabelic, Ph.D., Associate Professor, Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb

No.: #103788

Datum: 17.06.2019

Antigen: LGALS3

Chargennummer: 158844928901

Validierte Anwendung: Haemagglutination

Positivkontrolle:

human erythrocytes (blood group A)

serum from uncompatible donor (blood group B)

Negativkontrolle:

PBS

0.5mM lactose

Bewertung:

Passed. The recombinant human galectin-3 ABIN5509728 possesses lectin activity.

Validierungsbilder

Hemagglutination assay using recombinant human galectin-3 ABIN5509728. A. Plate Layout. B. and C. Pictures captured after 1h and 24h respectively.

Protokoll

Full Protocol:

• Isolate human erythrocytes and prepare trypsinized,glutaraldehyde-fixed erythrocytes
• • Take blood by venipuncture from healthy volunteer (blood type A) in one lavender vacutainer tube (containing EDTA as anticoagulant) and centrifuged at 500×g for 5min at 4°C.
  • Remove plasma and white cell ghost layer at the top of the pellet.
  • Resuspend red blood cells in 10ml cold PBS by gently aspirating and expelling them with a pipette. Centrifuge red blood cells at 500×g for 5min at 4°C. Remove the supernatant and repeat 3x.
  • Resuspend the pellet in 20ml PBS (Gibco) containing 0.05% trypsin and incubate at 37°C for 1h with an occasional shake.
  • Centrifuge at 500xg for 5min at 4°C.
  • Remove supernatant and resuspend erythrocytes in 10ml cold PBS.
  • Wash pellet 3x as described above.
  • Resuspend erythrocytes in 25ml PBS containing 1% glutaraldehyde and incubate for 1h at RT with continuous gentle shaking.
  • Centrifuge at 500xg for 5min at 4°C.
  • Remove supernatant and wash erythrocytes 2x with 0.1M glycine in PBS.
  • Wash erythrocytes with PBS.
  • Store erythrocytes as 4% (v/v) erythrocyte suspension at 4 °C until use (within one month of preparation).
• Isolation of human serum (blood type B)
• • Take blood by venipuncture from healthy volunteer (blood type B) in one red topped vacutainer tube (additive-free vacutainer).
  • Allow the blood to clot by leaving it undisturbed at room temperature for 30min.
  • Centrifuge at 1000xg for 10min at 4°C.
  • Remove serum supernatant and store at -20°C until use.
• Preparation of galectin-3 and lactose
• • Reconstitute lyophilized galectin-3 (antibodies-online, ABIN5509728, lot 0158844928901) in sterile, 0.2μm filtered solution of 0.2% bovine serum albumin (BSA) in PBS, according to the manufacturers’ recommendations, to 100μg/ml.
  • Dilute D-lactose in PBS to 2mM.
• Measurement of Hemagglutination
• • Prepare samples in triplicates as indicated in Figure A in a total volume of 100µl in a microtiter V plate.
  • Incubate plate for 1h and 24h at 4°C.
  • Determine results by visual examination. Image acquisition on an Amersham Imager 600, Image capturing method colorimetric-transillumination.

Anmerkungen:

• Galectin-3 is approximately 30kDa and, like all galectins, contains a carbohydrate-recognition-binding domain (CRD) of about 130 amino acids that enable the specific binding of β-galactosides. For several decades, hemagglutination assay has been used to obtain semi-quantitative data on the sugar binding and specificity of a particular lectin. Active lectins agglutinate erythrocytes by recognizing carbohydrates on the cell surface and forming a cross-linked network in suspension.

• By serially diluting the lectin in a 96-well microtiter plate and adding a constant quantity of erythrocytes, the lectin activity can be estimated. Galectin-3 lectin activity is abolished by the addition of lactose, which binds to its CRD domain and consequently inhibits hemagglutination. The assay is usually performed on trypsinized, glutaraldehyde-fixed red blood cells because they are more sensitive to hemagglutination reactions. Addition of bovine serum albumin (BSA) to all wells prevents non-specific interactions and consequently non-specific auto-hemagglutination. As positive control, serum of a non-compatible blood type can be used. Negative control is PBS. Control for abolishing galectin-3 activity is lactose.

• The hemagglutination assay is carried out by incubating human erythrocytes (blood group A or B) in the presence of increasing concentrations of galectin-3 in a microtiter V plate for 1h and 24h at RT in a final volume of 100µl. Galectin-3 recognizes glycans on both type A and type B red blood cells at submicromolar concentrations, according to the literature data, but does not bind to blood group O(H) at these concentrations. Commercially available 4% solutions of trypsinized, glutaraldehyde-fixed erythrocytes from various species and (e.g. rabbit) can also been used.

• Pre-incubation of galectin-3 for 30min in the presence of 500µM lactose should cause prevention (inhibition) of hemagglutination.

• Hemagglutination was detected in wells that contained the positive control without (wells A4-A6) and with lactose (wells A10-12), and in wells with galectin-3 at final concentrations 25μg/mL, 12.5μg/mL, and 5μg/mL (wells B1-9). A galectin-3 concentration of 1μg/ml was insufficient to cause complete hemagglutination (wells B10 to B12). Partial inhibition was detected in wells containing galectin-3 at 25µg/ml in the presence of 0.5mM lactose (well C1-3). At galectin-3 concentrations 12.5µg/ml, 5µg/ml, and 1µg/ml hemagglutination is completely abolished in the presence of 0.5mM lactose (well C4-12).

• References:

• https://www.ncbi.nlm.nih.gov/pubmed/1259723

• https://www.ncbi.nlm.nih.gov/pubmed/10722666

• https://www.ncbi.nlm.nih.gov/pubmed/22497898

• https://www.ncbi.nlm.nih.gov/pubmed/27328612

• https://www.ncbi.nlm.nih.gov/pubmed/30797745

• https://www.ncbi.nlm.nih.gov/pubmed/18216021

Handhabung

Format: Lyophilized

Rekonstitution: A quick spin of the vial followed by reconstitution in sterile distilled water to a concentration not less than 0.1 mg/mL is recommended. Please note, filter sterilization is a must following reconstitution. This solution can then be diluted into other buffers.

Lagerung: 4 °C,-20 °C

Informationen zur Lagerung: The lyophilized protein is stable for at least one year from date of receipt at -70°C. Upon reconstitution, this cytokine can be stored in working aliquots at 2° - 8°C for one month, or at -20°C for six months, with a carrier protein without detectable loss of activity. Avoid repeated freeze/thaw cycles.

Antigendetails

Target: Galectin 3 (LGALS3)

Andere Bezeichnung: Galectin-3 (LGALS3) (LGALS3 Produkte)

Synonyme: CBP 35 Protein, Gal-3 Protein, Galectin-3 Protein, leg3 Protein, galactoside-binding Protein, galectin-3 Protein, lectin Protein, soluble Protein, CBP35 Protein, GAL3 Protein, GALBP Protein, GALIG Protein, L31 Protein, LGALS2 Protein, MAC2 Protein, MGC52947 Protein, MGC75803 Protein, LGALS3 Protein, gal-3 Protein, GBP Protein, L-34 Protein, Mac-2 Protein, gal3 Protein, galectin 3 Protein, Galectin-3 Protein, avian beta-defensin 3 Protein, lectin, galactoside-binding, soluble, 3 L homeolog Protein, lectin, galactoside-binding, soluble, 3 Protein, galectin 2 Protein, lectin, galactose binding, soluble 3 Protein, LGALS3 Protein, leg3 Protein, AvBD3 Protein, lgals3.L Protein, lgals3 Protein, LGALS2 Protein, Lgals3 Protein

Substanzklasse: Chemical

Molekulargewicht: 26,1

Gen-ID: 3958

UniProt: P17931

Pathways: RTK Signalweg