Lens culinaris agglutinin lca

Manufactured by Vector Laboratories

Sourced in United States

Lens culinaris agglutinin (LCA) is a lectin derived from the seeds of the common lentil (Lens culinaris). It is a carbohydrate-binding protein that exhibits binding specificity for terminal α-D-mannose and α-D-glucose residues.

Automatically generated - may contain errors

Lab products found in correlation

Streptavidin horseradish peroxidase, by Vector Laboratories (2 mentions) Pvdf membrane, by Vector Laboratories (2 mentions) Western blot kit, by Vector Laboratories (2 mentions) Pvdf membrane, by Merck Group (2 mentions) Ulex europaeus agglutinin, by Vector Laboratories (2 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions) Pngase f, by New England Biolabs (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Bx51 fluorescent microscope, by Olympus (1 mentions) Hrp conjugated streptavidin, by Beyotime (1 mentions) Bca protein concentration determination kit, by Takara Bio (1 mentions) Ecl kit, by Beyotime (1 mentions) Anti αv, by Santa Cruz Biotechnology (1 mentions) Lotus tetragonolobus lectin, by Vector Laboratories (1 mentions) Celltracker green cmfda, by Thermo Fisher Scientific (1 mentions) Anti β3, by Santa Cruz Biotechnology (1 mentions) Multimode plate reader, by PerkinElmer (1 mentions) Mouse igg, by Santa Cruz Biotechnology (1 mentions) Fluorescent microscope, by Olympus (1 mentions) Nucblue live readyprobe, by Thermo Fisher Scientific (1 mentions)

9 protocols using lens culinaris agglutinin lca

Glycoengineered Immunoconjugate Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

A glycoengineered variant of the hSC16 immunoconjugate was prepared via enzymatic deglycosylation using 1 unit of PNGaseF (New England Biolabs) per microgram of DFO-conjugated hSC16 antibody (DFO-hSC16). 5 micrograms of the following antibody constructs – an isotype-matched anti-hapten humanized antibody (hIgG)(23 , 24 ) (provided by Abbvie Stemcentrx LLC), hSC16, DFO-hSC16, deglycosylated DFO-hSC16, and DFO-conjugated Fc-silent variant of hSC16 (DFO-hSC16_Fc-silent) – were electrophoresed on a NuPAGE 4–12% Bis-Tris gel (Thermo Fisher Scientific). The gel was stained with Coomassie blue to observe a shift in the migration of heavy chains in the deglycosylated and Fc-silent hSC16 immunoconjugates. A Western blot was performed and the nitrocellulose membrane was stained with Ponceau-S to confirm the successful transfer of antibody heavy chains from the gel. Finally, a carbohydrate analysis was performed using lens culinaris agglutinin (LCA; Vector Laboratories) to establish the presence versus absence of glycans in the various antibody constructs (seeSIfor details).

Sharma S.K., Chow A., Monette S., Vivier D., Pourat J., Edwards K.J., Dilling T.R., Abdel-Atti D., Zeglis B.M., Poirier J.T, & Lewis J.S. (2018). Fc-mediated Anomalous Biodistribution of Therapeutic Antibodies in Immunodeficient Mouse Models. Cancer research, 78(7), 1820-1832.

+ Open protocol

+ Expand

Glycan Profiling of Cryosections

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cryosections on coverslips were washed in buffer and blocked with a solution containing 3% of BSA/0.2% glycine in 0.1 M HEPES for 2 hours at RT, and incubated with biotinylated Lens culinaris agglutinin (LCA; Vector Laboratories) at a working concentration of 25 μg/ml. Rabbit polyclonal anti-α1,3-linked core-fucose serum was applied at a concentration of 3 μg/mL (Agrisera). All probes were diluted in 0.5% BSA and incubated with the sections for 2 hours. After washing with 0.5% BSA in HEPES, immunostaining was detected with Extravidin—fluorescein isothiocyanate (FITC) (Sigma-Aldrich, 15 μg/ml) and anti-rabbit Fab´IgG conjugated to Q dot 605 (QDs, Molecular Probes, 20 nM) for 1 h. Negative control experiments were performed by omitting the glycan-binding probes and specificity of glycan-binding probes was tested as described in the part supporting information (S1 Protocol, S2 Fig and S1 Table). The sections were washed in HEPES and stained in DAPI (Sigma-Aldrich, 0.01 μg/ml, 5 min). After rinsing, the coverslips with section side down were placed on buffer. Fluorescence was examined using an Olympus BX 51 fluorescent microscope. Immediately after the observations, the coverslips were placed section side down on buffer.

Vancová M, & Nebesářová J. (2015). Correlative Fluorescence and Scanning Electron Microscopy of Labelled Core Fucosylated Glycans Using Cryosections Mounted on Carbon-Patterned Glass Slides. PLoS ONE, 10(12), e0145034.

+ Open protocol

+ Expand

Protein Extraction and Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

The cervical exfoliated cells suspension was firstly centrifuged at 5000 rpm for 5 min. Add an appropriate amount of protein lysis solution to the cell pellet and lyse it on ice for 15–30 min. Gently shake the mixture during the lysis process to make it fully lysed. After lysis, centrifuge at 12,000 rpm for 10 min, collect the supernatant, use the BCA protein concentration determination kit (Takara Bio, Otsu, Japan) to determine the protein concentration of each sample. For serum samples, directly dilute 50 times by PBS and use BCA method to detect the protein concentration.
The total proteins in serum and cell samples were separated by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and were then transfered to the nitrocellulose filter (NC) membrane (Merck Millipore, USA). The membrane was sealed for 1 h at RT using 5% BSA and was subsequently incubated overnight at 4 °C with different primary antibodies or Lens Culinaris Agglutinin (LCA, Vector Laboratories). After that, the membrane was incubated with the HRP-conjugated streptavidin or HRP-conjugated Abs (Beyotime) at room temperature (RT) for 1 h, and the protein bands were then visualized by using the ECL kit (Beyotime).

Fan Q., Wu Y., Li M., An F., Yao L., Wang M., Wang X., Yuan J., Jiang K., Li W, & Li M. (2021). Lactobacillus spp. create a protective micro-ecological environment through regulating the core fucosylation of vaginal epithelial cells against cervical cancer. Cell Death & Disease, 12(12), 1094.

+ Open protocol

+ Expand

Adhesion Assay of JAR Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

HEC-1A, Ishikawa and RL95-2 cells were grown on 96-well plates, and treated with rhPAPPA, anti-PAPPA (Santa Cruz, USA), different human serum samples or different conditional media of JAR cells. After cells formed a confluent monolayer, mouse IgG, anti-αV, anti-β3, anti-αVβ3 (Santa Cruz, USA); Three Lectins: Ulex europaeus agglutinin (UEA-1), Lotus tetragonolobus lectin (LTL) and Lensculinaris agglutinin (LCA) (Vector Laboratories, USA) were added to block the specific epitope for 4 h. JAR cells were stained with CellTracker™ Green CMFDA (Invitrogen, USA) for 1 h before the adhesion assay. The stained cells (1 × 10⁴) were plated onto treated HEC-1A, Ishikawa or RL95-2 cell monolayers in JAR cell culture medium. After 1 h, unattached JAR cells were removed, and the attached cells were gently washed with PBS 3 times. The cells were than photographed under a fluorescent microscope (Olympus, Japan). An equal amount of stained JAR cells (1 × 10⁴) was plated in 3 blank wells. After detection using a multimode plate reader (PerkinElmer, USA), adhesion rate was calculated as a percentage of attached JAR cells. All experiments were replicated 3 times.

Yu M., Wang J., Liu S., Wang X, & Yan Q. (2017). Novel function of pregnancy-associated plasma protein A: promotes endometrium receptivity by up-regulating N-fucosylation. Scientific Reports, 7, 5315.

+ Open protocol

+ Expand

Selection and Identification of FUT8 Knockout Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Five days after transfection (Day 5), selection of FUT8 knockout cells was initiated by supplementing complete medium with 50 µg/mL Lens culinaris agglutinin (LCA; Vector Laboratories, Peterborough, UK) from a 5 mg/mL LCA (10 mM Hepes/NaOH, pH 8.5, 0.15 mM NaCl, 0.1 mM CaCl₂) stock solution. Bright field images were taken with a Celigo Imaging Cell Cytometer (Brooks Automation). After 7 days of selection (Day 12), genomic DNA was extracted as described above. In parallel, cells were seeded in complete medium without LCA. The day after (Day 13), cells were incubated for 45 min at RT in complete medium containing 20 µg/mL fluorescein-LCA (Vector Laboratories) and two droplets NucBlue® Live ReadyProbes (Life Technologies) per mL media. Cells were washed three times with complete medium and fluorescence microscopy was performed on a LEAP instrument (Intrexon, Germantown, MD) using the two channel imaging application with the NucBlue stain as target 1 using the blue fluorescence channel and the fluorescein labeled LCA as target 2 using the green fluorescence channel.

Ronda C., Pedersen L.E., Hansen H.G., Kallehauge T.B., Betenbaugh M.J., Nielsen A.T, & Kildegaard H.F. (2014). Accelerating Genome Editing in CHO Cells Using CRISPR Cas9 and CRISPy, a Web-Based Target Finding Tool. Biotechnology and Bioengineering, 111(8), 1604-1616.

+ Open protocol

+ Expand

Biotinylated Lectins for Fucose Detection

Check if the same lab product or an alternative is used in the 5 most similar protocols

Three biotinylated fucose-specific lectins: Lotus tetragonolobus agglutinin (LTA, catalog No. B-1325, Vector Laboratories Inc., Burlingame, CA, USA), Ulex europaeus agglutinin (UEA, catalog No. B-1065, Vector Laboratories Inc., Burlingame, CA, USA) and Lens culinaris agglutinin (LCA, catalog No. B-1045, Vector Laboratories Inc., Burlingame, CA, USA) were used to determine fucose expression in the lectin-ELISA procedure according to the Kratz et al.²⁴ with modifications described below. The specificity of lectins is not absolute, and they can react with more than one oligosaccharide residue. Lotus tetragonolobus agglutinin and Ulex europaeus agglutinin detect fucoses linked to the galactose or antennary N-acetylglucosamine by α1,3 glycosidic bond and α1,2 glycosidic bond, respectively^{28 (link)}. Lens culinaris agglutinin was used for core fucose detection^{29 (link)}.

Janiszewska E., Kokot I., Gilowska I., Faundez R, & Kratz E.M. (2021). The possible association of clusterin fucosylation changes with male fertility disorders. Scientific Reports, 11, 15674.

+ Open protocol

+ Expand

Lens Culinaris Agglutinin Glycoprotein Detection

Check if the same lab product or an alternative is used in the 5 most similar protocols

The CRC cells were lysed and separated via 10% SDS-PAGE and transferred to polyvinylidene di uoride (PVDF) membranes (Millipore, Bedford, MA, USA). Then the PVDF membranes were incubated with the biotinylated Lens Culinaris Agglutinin (LCA) (Vector Laboratories, Burlingame, CA, USA) for 1h at room temperature. The membranes were subsequently incubated with horseradish peroxidase streptavidin (Vector Laboratories) for 30 min and detected with an ECL Western blot kit.

Qi Y., Shan Y., Li S., Huang Y., Guo Y., Huang T., Zhao X, & Jia L. (2022). LncRNA LEF1-AS1/LEF1/FUT8 Axis Mediates Colorectal Cancer Progression by Regulating α1, 6-Fucosylationvia Wnt/β-Catenin Pathway. Digestive diseases and sciences, 67(6).

+ Open protocol

+ Expand

Clonal Selection of Stably Transfected Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were stably transfected with different donor plasmids and/or sgRNA plasmids. For CHO-K1 cells, 8 μg/mL of puromycin (Amresco, Solon, OH, USA) was used for 2 weeks and 50 μg/mL Lens culinaris agglutinin (LCA) (Vector Laboratories, Peterborough, UK) for an additional week to enrich integrated cells. For CHO-S cells, 8 μg/mL puromycin was used for 3 weeks. Stably transfected pools were then detected with 5′/3′ junction PCR and Western blot, and further seeded at 0.5-1 cell/well in 96-well plates for limiting dilution. After another 3-4 weeks, the colonies were generated and analyzed by 5′/3′ junction PCR and Western blot for protein expression. Only those colonies positive in both junction PCR and Western blot assessment were selected for further investigation.
Genomic DNA extraction and 5′/3′ junction PCR at target region Genomic DNA was extracted with QuickExtract DNA extraction solution (Epicenter, Madison, WI, USA) from stably transfected pools and single cell clones for the amplification of 5′/3′ junction PCR according to the following PCR procedure: 94 °C for 2 min; 30×: 94 °C for 30 s, 65 °C-50 °C (-0.5 °C/cycle) for 30 s, 72 °C for 2 min; 20×: 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 2 min. Primers are listed in Supplementary Table S1. Purified 5′/3′ junction PCR product was sequenced by Invitrogen.

Zhao M., Wang J., Luo M., Luo H., Zhao M., Han L., Zhang M., Yang H., Xie Y., Jiang H., Feng L., Lu H., & Zhu J. (2018). Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35. Applied Microbiology and Biotechnology, 102(14), 6105-6117.

+ Open protocol

+ Expand

Lens Culinaris Agglutinin Glycoprotein Detection

Check if the same lab product or an alternative is used in the 5 most similar protocols

Qi Y., Shan Y., Li S., Huang Y., Guo Y., Huang T., & Li J. (2020). LncRNA LEF1-AS1/LEF1/FUT8 Axis Mediates Colorectal Cancer Progression by Regulating α1, 6-Fucosylation via Wnt/β-Catenin Pathway.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!