CN34 tumour cells were isolated from the pleural effusion of a breast cancer patient treated at our institution, after written consent in accordance with Institutional Review Board (IRB) regulations. Brain metastatic populations from these cells and MDA-MB-231cells were obtained by consecutive rounds of in vivo selection in 6–7-week-old beige nude and athymic mice, respectively. All animal work was done in accordance with the MSKCC Institutional Animal Care and Use Committee. Methods for RNA extraction, labelling and hybridization for DNA microarray analysis have been described previously17 (link). Bioinformatics analyses with detailed descriptions can be found in the Methods. Knockdown and overexpression of candidate genes, and cetuximab inhibitor studies were performed as previously described6 (link). The in vitro BBB model was set up as previously described25 (link), and modified to enable tumour cell counting. Sambucus nigra lectin staining was performed using standard histochemical techniques, and quantified using Metamorph software analysis. The Methods section provides further information, including malignant cell isolation from pleural fluids, tumour cell extraction and cell culture protocols, animal inoculation and bioluminescence imaging, generation of retroviral gene knockdown and overexpression vectors, transfections and infections, RNA and protein expression, in vitro BBB transmigration assay, endothelial cell adhesion assay, and metastatic tissue staining and quantification.
>
Chemicals & Drugs
>
Amino Acid
>
Lectin
Lectin
Lectins are a diverse class of carbohydrate-binding proteins found in many organisms, including plants, animals, and microbes.
These proteins play crucial roles in various biological processes, such as cell-cell recognition, signaling, and immune response.
Lectins can bind to specific sugar moieties on the surface of cells, making them valuable tools for researchers studying cell surface interactions and glycobiology.
Exploring the properties and applications of lectins is a key focus of lectin research, with potential implications in areas like cancer diagnostics, therapeutics, and glycoprotein analysis.
PubCompare.ai can help streamline your lectin studies by identifying the most reproducibel and reliable methods from the scientific literature, pre-prints, and patents.
These proteins play crucial roles in various biological processes, such as cell-cell recognition, signaling, and immune response.
Lectins can bind to specific sugar moieties on the surface of cells, making them valuable tools for researchers studying cell surface interactions and glycobiology.
Exploring the properties and applications of lectins is a key focus of lectin research, with potential implications in areas like cancer diagnostics, therapeutics, and glycoprotein analysis.
PubCompare.ai can help streamline your lectin studies by identifying the most reproducibel and reliable methods from the scientific literature, pre-prints, and patents.
Most cited protocols related to «Lectin»
Animals
Biological Assay
Brain
Breast Carcinoma
Cell Adhesion
Cell Culture Techniques
Cells
Cell Separation
Cetuximab
Cloning Vectors
Crossbreeding
DNA Chips
Endothelial Cells
Endothelium
Ethics Committees, Research
Gene Knockdown Techniques
Genes
Infection
Institutional Animal Care and Use Committees
Lectin
Mice, Nude
Microarray Analysis
Neoplasms
Patients
Pleura
Pleural Effusion
Population Group
Proteins
Retroviridae
Sambucus nigra
Tissues
Transfection
Vaccination
Antibodies
Antibodies, Anti-Idiotypic
Astrocytes
Brain
Cell Culture Techniques
Cells
Endothelial Cells
Fetus
Gray Matter
Homo sapiens
Hybridomas
Hyperostosis, Diffuse Idiopathic Skeletal
Lectin
Lysine
Macrophage
Microglia
Neurons
Oligodendrocyte Precursor Cells
Oligodendroglia
Papain
Poly A
Protease Inhibitors
RNA-Seq
Serum
Thy-1 Antigens
Tissues
Trypsin
After myocardial infarction and four weeks of reperfusion, hearts were either paraffin embedded or the raw material was snap frozen at -40°C in isopentane. In paraffin sections (4 µm) the wax was dissolved by an organic solvent and the tissue slices rehydrated before picrosirius red stain was applied. Cryosections (8 µm) from raw snap frozen tissue were fixed in 4% paraformaldehyde (PFA) in 0.1M sodium phosphate buffer (PB) pH 7.4 or Zambonis fixative (0.1 M PB, 4% (w/v) PFA, 15% (v/v) picric acid) for 10 min. Washing steps were performed in PBS, PBS/0.1% Saponin or in PBS/0.2% Tween 20 according to the further requirements. Picrosirius red (SR) staining was performed according to the protocols of Junqueira et al.3 (link) and Sweat et al.5 (link)Wheat germ agglutinin (WGA) labeling was routinely used in combination with the secondary antibody in immunohistochemical preparations. Lectin from triticum vulgaris FITC conjugate (# L4895, Sigma-Aldrich, St. Louis, MO, USA) was diluted 1:100 (10 µg/mL) in the required buffer. Incubation time was one hour protected from light. After three washing steps, the sections were coverslipped with a water-soluble antifading mounting medium. Collagen I staining was performed using an anti-collagen I antibody (ab34710, Abcam, Cambridge, UK) diluted 1:100 in the required buffer. As secondary antibody anti-Rabbit-Cy3 (111-165-144, Jackson Dianova, Hamburg, Germany) was used in a concentration of 2.5 µg/mL.
Slides were analyzed with fluorescence microscope Keyence BZ 9000 (Keyence, NeuIsenburg, Germany). All shown images were taken with 4x objective using the merge function, as not otherwise specified.
Slides were analyzed with fluorescence microscope Keyence BZ 9000 (Keyence, NeuIsenburg, Germany). All shown images were taken with 4x objective using the merge function, as not otherwise specified.
Antibodies, Anti-Idiotypic
Buffers
Collagen Type I
Cryoultramicrotomy
Fixatives
Fluorescein-5-isothiocyanate
Freezing
Heart
Immunoglobulins
isopentane
Lectin
Light
Microscopy, Fluorescence
Myocardial Infarction
Paraffin
paraform
Phosphates
picric acid
Rabbits
Reperfusion
Saponin
sodium phosphate
Solvents
Stains
Sweat
Tissues
Triticum
Tween 20
Wheat Germ Agglutinins
Human FcγRs (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb) and MBL were produced via transient transfection in HEK293 cells, and purified via immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography (SEC) as described previously (Boesch et al., 2014 (link)). Size and purity of all recombinant proteins was confirmed by SDS-PAGE. Human C1q (Fischer Scientific ICN19139101) was purchased unlabeled and biotinylated according to the procedure described below. Biotinylated lectin detection reagents (SNA, ConA, GNL, MAL, LCA, RCA, PNA, AAL, VVL, and UAE1) were purchased from Vector Laboratories (B-1305, B-1005, B-1045, B-1245, B-1085, B-1075, B-1235, B-1315, B-1065, B-1395).
FcRs were chemically biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (Pierce 21331) at a molar ratio of 5 mols biotin per mol of protein. Biotinylation was carried out for 2 h at RT, with a protein concentration of 0.2 mg/mL. Afterwards, excess biotin was removed via 3 rounds of dilution with PBS and concentration using 3 kD cutoff centrifugal filter units (Amicon UFC900396).
Immediately prior to use, the biotinylated FcR was mixed with a 1/4th molar ratio of Streptavadin-PE (Prozyme PJ31S), diluted to a final concentration of 1.0 μg/mL FcγR in Assay Buffer (PBS-1X + 0.1% BSA + 0.05% Tween20), and mixed for 10 min with rotation. After mixing, 1% v/v of 500 μM free biotin was added to completely block any free streptavidin binding sites. Biotinylated lectin reagents were obtained commercially from Vector Laboratories. Lectin tetramers were produced in the same manner as FcRs, except that the dilution buffer was 20 mM Tris pH 8.0 + 0.1 mM Ca ++, Mg ++, Mn ++.
FcRs were chemically biotinylated using EZ-Link Sulfo-NHS-SS-Biotin (Pierce 21331) at a molar ratio of 5 mols biotin per mol of protein. Biotinylation was carried out for 2 h at RT, with a protein concentration of 0.2 mg/mL. Afterwards, excess biotin was removed via 3 rounds of dilution with PBS and concentration using 3 kD cutoff centrifugal filter units (Amicon UFC900396).
Immediately prior to use, the biotinylated FcR was mixed with a 1/4th molar ratio of Streptavadin-PE (Prozyme PJ31S), diluted to a final concentration of 1.0 μg/mL FcγR in Assay Buffer (PBS-1X + 0.1% BSA + 0.05% Tween20), and mixed for 10 min with rotation. After mixing, 1% v/v of 500 μM free biotin was added to completely block any free streptavidin binding sites. Biotinylated lectin reagents were obtained commercially from Vector Laboratories. Lectin tetramers were produced in the same manner as FcRs, except that the dilution buffer was 20 mM Tris pH 8.0 + 0.1 mM Ca ++, Mg ++, Mn ++.
Full text: Click here
Binding Sites
Biological Assay
Biotin
biotin 1
Biotinylation
Buffers
Cardiac Arrest
Chromatography, Affinity
Cloning Vectors
Concanavalin A
Fc gamma receptor IIA
Fc Receptor
Gel Chromatography
HEK293 Cells
Homo sapiens
Lectin
Metals
Molar
Proteins
Recombinant Proteins
SDS-PAGE
Staphylococcal Protein A
Streptavidin
sulfo-N-hydroxysuccinimide-biotin
Technique, Dilution
Tetrameres
Transfection
Transients
Tromethamine
Tween 20
Antibodies
ARID1A protein, human
Biological Assay
carbohydrate-binding protein
Cells
Disulfides
Dithiothreitol
Electrophoresis
Genetic Heterogeneity
Hydrochloride, Guanidine
Lectin
Light Microscopy
Microscopy
Mucins
Polymers
Protein Glycosylation
Protoplasm
secretion
Transmission Electron Microscopy
Vacuum
Most recents protocols related to «Lectin»
Example 4
An overview of the immunization strategies for lectin-binding proteins, such as galectin-3, is shown in Table 18.
BALB/c mice were immunized with 2 mg/kg mRNA, complexed with LNPs, or 20 μg recombinant protein as indicated in Table 18. Plasma anti-galectin-3 IgG titers were assayed 7 days after the final boost, which was delivered at day 55.
Hybridomas producing galectin-3-specific antibodies were generated, and high affinity monoclonal anti-galectin-3 antibodies were obtained from further screens.
Table 19 provides a target protein-specific summary of the total number of hybridoma wells (generally about one third (⅓) of these wells contain hybridomas) screened and the number of confirmed target-specific antibodies obtained from those hybridomas wells following the use of lipid-encapsulated mRNA as an immunogen.
Table 20 provides a comparison of mRNA-LNP immunization methods with other conventional methods of immunization by number of hybridomas producing target-specific antibodies. In general, these data suggest that mRNA-LNP immunization is an effective method for inducing an immune response to a target protein antigen and for obtaining a higher number/rate of target protein-specific antibodies. In particular, these results confirm that mRNA-LNP immunization is surprisingly more effective than conventional immunization methods for obtaining antibodies specific for transmembrane proteins, e.g., multi-pass transmembrane proteins, such as GPCRs, which are difficult to raise antibodies against, and for poorly immunogenic proteins (e.g., proteins which produce low or no detectable target-specific IgGs in plasma of animals immunized with traditional antigen).
In general, successful generation of hybridomas producing antigen-specific antibodies have been achieved for at least 15 different targets utilizing mRNA-LNP immunization methods as exemplified herein. These results show that the mRNA immunization methods described herein are capable of eliciting an immune response against a wide range of antigens (e.g., transmembrane proteins, for example multi-pass transmembrane proteins, such as GPCRs) in host animals, and are effective methods for producing high affinity monoclonal antibodies, which can serve as parentals for generation of chimeric variants, humanized variants, and affinity matured variants.
Full text: Click here
Animals
anti-IgG
Antibodies
Antigens
Binding Proteins
Cells
Chimera
DNA, Complementary
Epitopes
Galectin 3
Histocompatibility Antigens Class II
Homo sapiens
Hybridomas
Integral Membrane Proteins
Lectin
Lipids
Mice, Inbred BALB C
Monoclonal Antibodies
Parent
Peptides
Plasma
Proteins
Protein Targeting, Cellular
Recombinant Proteins
Response, Immune
RNA, Messenger
Soluble Glycoprotein 130
Thyroid Gland
Thyrotropin
Thyrotropin Receptor
Vaccination
Viral Proteins
Example 2
Comparison in Number of ABA/ACA-Specific Exosomes Between Pancreatic Cancer and Other Cancer Types
It was studied whether there was a difference in the amount of ABA/ACA-specific exosomes between cancer types from healthy subjects' sera and sera from each of pancreatic cancer patients, esophageal cancer patients, and colorectal cancer patients. The quantitative determination of ABA/ACA-specific exosomes was performed through the method described in (Method for Measuring Exosomes Binding to Lectins) described above.
The results are shown in
Full text: Click here
Colorectal Carcinoma
Esophageal Cancer
Exosomes
Healthy Volunteers
Lectin
Malignant Neoplasms
Pancreatic Cancer
Patients
Serum
Example 1
Comparison in Number of ABA/ACA-Specific Exosomes Between Healthy Subject and Pancreatic Cancer Patient
It was studied whether there was a difference in the amount of ABA/ACA-specific exosomes between healthy subjects' sera and preoperative and postoperative sera from pancreatic cancer patients. The quantitative determination of ABA/ACA-specific exosomes was performed through the method described in (Method for Measuring Exosomes Binding to Lectins) described above. The sera were diluted 4 times with PBS-T before use.
The results are shown in
Full text: Click here
Exosomes
Healthy Volunteers
Lectin
Operative Surgical Procedures
Pancreatic Cancer
Patients
Recurrence
Serum
Protocol full text hidden due to copyright restrictions
Open the protocol to access the free full text link
Base Sequence
Cloning Vectors
Exons
Figs
Hexosaminidase A
Histidine
Lectin
Recombination, Genetic
Sequence Alignment
Protocol full text hidden due to copyright restrictions
Open the protocol to access the free full text link
Cytokinesis
Lectin
Proteolysis
SDS-PAGE
SERPINA1 protein, human
Tromethamine
Trypsin
Top products related to «Lectin»
Sourced in United States, Germany, United Kingdom, China, Italy, Japan, France, Sao Tome and Principe, Canada, Macao, Spain, Switzerland, Australia, India, Israel, Belgium, Poland, Sweden, Denmark, Ireland, Hungary, Netherlands, Czechia, Brazil, Austria, Singapore, Portugal, Panama, Chile, Senegal, Morocco, Slovenia, New Zealand, Finland, Thailand, Uruguay, Argentina, Saudi Arabia, Romania, Greece, Mexico
Bovine serum albumin (BSA) is a common laboratory reagent derived from bovine blood plasma. It is a protein that serves as a stabilizer and blocking agent in various biochemical and immunological applications. BSA is widely used to maintain the activity and solubility of enzymes, proteins, and other biomolecules in experimental settings.
Sourced in United States, United Kingdom, Germany, Japan, France, Italy, Canada, China, Spain, Switzerland, Denmark, Australia, Hungary, Belgium, Ireland, Israel, Netherlands, Moldova, Republic of, India, Austria, Czechia, Poland
Alexa Fluor 488 is a fluorescent dye used in various biotechnological applications. It has an excitation maximum at 495 nm and an emission maximum at 519 nm, producing a green fluorescent signal. Alexa Fluor 488 is known for its brightness, photostability, and pH-insensitivity, making it a popular choice for labeling biomolecules in biological research.
Sourced in United States
Biotinylated lectins are a class of glycan-binding proteins derived from plants, animals, and microorganisms. They are covalently labeled with biotin, a small molecule that can be used to attach the lectin to various substrates or detection systems. Biotinylated lectins are widely used in biochemical and cell biology applications to identify and study the distribution of specific carbohydrate structures on the surfaces of cells and in biological samples.
Sourced in United States, Canada
Carbo-Free Blocking Solution is a ready-to-use reagent designed to block non-specific binding in immunoassays. It is a protein-free, carbohydrate-based solution that effectively reduces background signal to enhance the specificity of target detection.
Sourced in United States, Germany, China, United Kingdom, Morocco, Ireland, France, Italy, Japan, Canada, Spain, Switzerland, New Zealand, India, Hong Kong, Sao Tome and Principe, Sweden, Netherlands, Australia, Belgium, Austria
PVDF membranes are a type of laboratory equipment used for a variety of applications. They are made from polyvinylidene fluoride (PVDF), a durable and chemically resistant material. PVDF membranes are known for their high mechanical strength, thermal stability, and resistance to a wide range of chemicals. They are commonly used in various filtration, separation, and analysis processes in scientific and research settings.
Sourced in United States, Germany, United Kingdom, China, Macao, Japan, Spain, Sao Tome and Principe, Denmark, France, Switzerland, Italy, Canada, Australia, India
Concanavalin A is a lectin protein derived from the jack bean plant. It is commonly used as a research tool in biochemistry and cell biology laboratories. Concanavalin A has the ability to agglutinate (bind and aggregate) certain types of cells, making it useful for studying cell-surface carbohydrates and their interactions.
Sourced in United States, Germany, United Kingdom, Japan, China, Canada, Italy, Australia, France, Switzerland, Spain, Belgium, Denmark, Panama, Poland, Singapore, Austria, Morocco, Netherlands, Sweden, Argentina, India, Finland, Pakistan, Cameroon, New Zealand
DAPI is a fluorescent dye used in microscopy and flow cytometry to stain cell nuclei. It binds strongly to the minor groove of double-stranded DNA, emitting blue fluorescence when excited by ultraviolet light.
Sourced in United States, Germany, Japan, United Kingdom, China, Italy, Sao Tome and Principe, France, Macao, Canada, Switzerland, Spain, Australia, Denmark, India, Poland, Israel, Belgium, Sweden, Ireland, Netherlands, Panama, Brazil, Portugal, Czechia, Puerto Rico, Austria, Hong Kong, Singapore
DAPI is a fluorescent dye that binds strongly to adenine-thymine (A-T) rich regions in DNA. It is commonly used as a nuclear counterstain in fluorescence microscopy to visualize and locate cell nuclei.
Sourced in United States, Germany, United Kingdom, Italy, China, Japan, France, Canada, Sao Tome and Principe, Switzerland, Macao, Poland, Spain, Australia, India, Belgium, Israel, Sweden, Ireland, Denmark, Brazil, Portugal, Panama, Netherlands, Hungary, Czechia, Austria, Norway, Slovakia, Singapore, Argentina, Mexico, Senegal
Triton X-100 is a non-ionic surfactant commonly used in various laboratory applications. It functions as a detergent and solubilizing agent, facilitating the solubilization and extraction of proteins and other biomolecules from biological samples.
Sourced in United States, United Kingdom, Germany, Japan, Canada, France, China, Morocco, Italy
PNGase F is an enzyme that cleaves the bond between the asparagine residue and the N-acetylglucosamine residue in N-linked glycoproteins. It is commonly used in the analysis and characterization of glycoproteins.
More about "Lectin"
Lectins are a diverse class of carbohydrate-binding proteins found in a variety of organisms, including plants, animals, and microbes.
These proteins, also known as agglutinins or hemagglutinins, play crucial roles in various biological processes such as cell-cell recognition, signaling, and immune response.
Lectins are capable of binding to specific sugar moieties (glycans) on the surface of cells, making them valuable tools for researchers studying cell surface interactions and glycobiology.
This property has led to the exploration of lectins' potential applications in areas like cancer diagnostics, therapeutics, and glycoprotein analysis.
Bovine serum albumin (BSA) is often used in conjunction with lectins to block non-specific binding, while Alexa Fluor 488 and biotinylated lectins are commonly employed as labeling agents for visualizing and detecting lectin-carbohydrate interactions.
Carbo-Free Blocking Solution can also be used to minimize background staining in lectin-based assays.
The versatility of lectins is further highlighted by their use in techniques like Western blotting, where PVDF membranes are often utilized to immobilize proteins for lectin-binding studies.
Concanavalin A, a well-known plant lectin, is frequently used as a tool for glycoprotein analysis and purification.
Nuclear staining dyes like DAPI can be used in combination with lectins to provide a comprehensive overview of cellular architecture and glycan distribution.
Triton X-100, a nonionic detergent, is commonly employed to permeabilize cells and enhance lectin accessibility to intracellular glycans.
Additionally, the enzyme PNGase F is often utilized to remove N-linked glycans from proteins, allowing researchers to study the effects of glycosylation on lectin binding and specificity.
PubCompare.ai can streamline your lectin studies by identifying the most reproducible and reliable methods from the scientific literature, preprints, and patents.
Leverage our AI-driven platform to optimize your lectin research and explore the diverse applications of these fascinating carbohydrate-binding proteins.
These proteins, also known as agglutinins or hemagglutinins, play crucial roles in various biological processes such as cell-cell recognition, signaling, and immune response.
Lectins are capable of binding to specific sugar moieties (glycans) on the surface of cells, making them valuable tools for researchers studying cell surface interactions and glycobiology.
This property has led to the exploration of lectins' potential applications in areas like cancer diagnostics, therapeutics, and glycoprotein analysis.
Bovine serum albumin (BSA) is often used in conjunction with lectins to block non-specific binding, while Alexa Fluor 488 and biotinylated lectins are commonly employed as labeling agents for visualizing and detecting lectin-carbohydrate interactions.
Carbo-Free Blocking Solution can also be used to minimize background staining in lectin-based assays.
The versatility of lectins is further highlighted by their use in techniques like Western blotting, where PVDF membranes are often utilized to immobilize proteins for lectin-binding studies.
Concanavalin A, a well-known plant lectin, is frequently used as a tool for glycoprotein analysis and purification.
Nuclear staining dyes like DAPI can be used in combination with lectins to provide a comprehensive overview of cellular architecture and glycan distribution.
Triton X-100, a nonionic detergent, is commonly employed to permeabilize cells and enhance lectin accessibility to intracellular glycans.
Additionally, the enzyme PNGase F is often utilized to remove N-linked glycans from proteins, allowing researchers to study the effects of glycosylation on lectin binding and specificity.
PubCompare.ai can streamline your lectin studies by identifying the most reproducible and reliable methods from the scientific literature, preprints, and patents.
Leverage our AI-driven platform to optimize your lectin research and explore the diverse applications of these fascinating carbohydrate-binding proteins.