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Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary:The cells targeted by the Nanobeads are either selected or depleted by incubating your sample with the directly conjugated magnetic particles. The magnetically labeled fraction is retained by the use of a magnetic separator. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc. Note: This procedure is optimized for the isolation of 107 to 2 x 108 cells per tube. If working with fewer than 107 cells, keep volumes as indicated for 107 cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water. Scale up volumes if using 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.

Proper citation: Sam Li 2019. MojoSort™ Mouse CD4 Nanobeads Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7wthpen Copy   

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Authors: Kelsey Knight
Group: BioLegend
Summary: The following is a general procedure guide for preparation and staining of acetone-fixed frozen tissues using a purified, unconjugated primary antibody, biotinylated secondary antibody and streptavidin-horseradish peroxidase (Sav-HRP) and DAB detection system. Because each antigen differs in terms of requirement for fixation, amplification step, etc., it is not possible to write an inclusive protocol that will work for all antigens. The user must determine optimal conditions for each antigen of interest. Many protocols for staining individual antigens, as well as useful tips and troubleshooting guides for immunohistochemistry, can be found at the IHC World web site (http://www.ihcworld.com/).

Proper citation: Kelsey Knight 2018. Immunohistochemistry Protocol for Frozen Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.tkfektn Copy   

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Authors: Sam Li
Group: BioLegend
Summary: Catalog Number: 658502Storage Temperature: 2°C-8°CProduct Description: Anti-c-Myc Tag (9E10) Affinity Gel consists of anti-c-myc monoclonal antibody (clone 9E10), covalently immobilized onto 6% high density glyoxal agarose beads. The affinity resin can be used in affinity purifying and immunoprecipitation of c-Myc-tagged fusion protein.Binding Specificity: Mouse monoclonal antibody 9E10 recognizes the c-myc epitope N-EQKLSEEDL-C and is purified through Protein G chromatography. The antibody conjugated affinity resin can bind to epitope at N-terminal, C-terminal, and internal locations of a fusion protein. The binding capacity is greater than 0.5mg per ml of c-myc agarose resin.Reagent: Anti-c-Myc Agarose Affinity Gel is supplied as a 50% suspension in phosphate-buffered saline, pH 7.2, containing 0.09% sodium azide.Precautions and Disclaimer: This product is for research use only. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.Storage/Stability: Anti-c-Myc Agarose Affinity Gel should be stored between 2°C and 8°C for maximum stability. The unopened product is stable for one year when stored as indicated. After use, the resin should be regenerated and stored in TBS or PBS buffer (pH 7.2) containing 0.09% sodium azide.

Proper citation: Sam Li 2019. Anti-c-Myc Tag (9E10) Affinity Gel Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.97ih9ke Copy   

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Authors: Kelsey Knight
Group: BioLegend

Proper citation: Kelsey Knight 2018. Th2 Polarization of Mouse CD4+ Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.tnkemcw Copy   

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Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ Nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing in academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads than with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.

Proper citation: Sam Li 2020. MojoSort™ Mouse Ig light chain κ Nanobeads Column Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bbueinte Copy   

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Authors: Kelsey Miller
Group: BioLegend

Proper citation: Kelsey Miller 2016. Alpha-Synuclein ELISA Kit Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.e34bgqw Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. Cell Surface Flow Cytometry Staining of Whole Blood. protocols.io https://dx.doi.org/10.17504/protocols.io.babbiain Copy   

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Authors: BioLegend, Inc.
Group: BioLegend

Proper citation: BioLegend, Inc. 2016. True-Nuclear™ Transcription Factor Staining Protocol for 5 mL Tubes. protocols.io https://dx.doi.org/10.17504/protocols.io.exrbfm6 Copy   

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Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.

Proper citation: Sam Li 2019. MojoSort™ Selection Kits Column Protocol - 5. protocols.io https://dx.doi.org/10.17504/protocols.io.7u3hnyn Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: Using UV-induced peptide exchange, MHC/peptide monomers can be generated with conditional Flex-T™ monomers that harbor peptides of interest in their binding grooves. These new MHC monomers are subsequently multimerized using streptavidin-fluorophore conjugates. The resulting Flex-T™ reagents can be used for staining antigen-specific T cells and flow cytometric analysis. In humans, the MHC molecules are called HLA (Human Leukocyte Antigen).

Proper citation: Kelsey Miller 2016. Flex-T™ Tetramer and Cell Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.e7bbhin Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. True-Nuclear™ Transcription Factor Staining Protocol for 5 mL Tubes. protocols.io https://dx.doi.org/10.17504/protocols.io.bacsiawe Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: The Anti-Neu5Gc Antibody Kit contains the essential monospecific polyclonal chicken IgY antibody, along with a negative control primary antibody to detect the presence of Neu5Gc on glycoconjugates by Western blot (WB). Samples to be evaluated are first subjected to SDS-PAGE, followed by transfer to a nitrocellulose or polyvinylidenedifluoride (PVDF) membrane. The membrane is then incubated with affinity-purified polyclonal anti-Neu5Gc to determine the presence of Neu5Gc on the protein of interest.The antibody provided in this kit has been shown to identify as little as 5 pmol of Neu5Gc per ug glycoprotein, which is at or below the current detection limit for conventional analysis by acid release, purification, DMB derivatization, HPLC, and electrospray mass-spectrometry. The Western blot provides additional information in that it confirms that Neu5G is directly linked to the glycoprotein of interest rather than to an accompanying sample component.

Proper citation: Kelsey Miller 2017. Anti-Neu5Gc Antibody Kit Protocol - Western Blot. protocols.io https://dx.doi.org/10.17504/protocols.io.hvrb656 Copy   

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Authors: Sam Li
Group: BioLegend
Summary: Using UV-induced peptide exchange, MHC/peptide monomers can be generated with conditional Flex-T™ monomers that harbor peptides of interest in their binding grooves. These new MHC monomers are subsequently multimerized using streptavidin-fluorophore conjugates. The resulting Flex-T™ reagents can be used for staining antigen-specific T cells and flow cytometric analysis. In humans, the MHC molecules are called HLA (Human Leukocyte Antigen).

Proper citation: Sam Li 2019. Flex-T™ Tetramer and Cell Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.babeiaje Copy   

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Authors: Kelsey Miller
Group: BioLegend

Proper citation: Kelsey Miller 2016. Propidium Iodide Cell Cycle Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.e2mbgc6 Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. Cell Surface Flow Cytometry Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.baa9iah6 Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: This protocol is essentially the same as a standard ELISA protocol, with one major exception – all animal compounds must be avoided in buffers, etc. because they contain Neu5Gc, which will interfere with antibody activity and result in misleading data. Essentially, the glycoconjugate to be evaluated is immobilized on an ELISA plate and exposed to a primary antibody. The antibody provided in this kit has been shown to identify as little as 5 pmol of Neu5Gc per microgram glycoprotein, which is at or below the current detection limit for conventional DMB HPLC analysis. An advantage of the ELISA procedure is that it confirms the presence of Neu5Gc on the glycoconjugate of interest, but use caution, as it also reacts with solution/media that contains glycoconjugate.

Proper citation: Kelsey Miller 2017. Anti-Neu5Gc Antibody Kit Protocol - ELISA. protocols.io https://dx.doi.org/10.17504/protocols.io.hvpb65n Copy   

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Authors: Kelsey Knight
Group: BioLegend

Proper citation: Kelsey Knight 2018. Cell Surface Immunofluorescence Staining Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.tihekb6 Copy   

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Authors: Kelsey Miller
Group: BioLegend
Summary: Target cells are depleted by incubating your sample with the biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads (Cat. No. 480015/480016). The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells are collected. These are your cells of interest; do not discard the liquid. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.

Proper citation: Kelsey Miller 2016. MojoSort™ Isolation Kits No Wash Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.e2wbgfe Copy   

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Authors: Sam Li
Group: BioLegend
Summary: T regulatory cells (also known as Tregs or Regulatory T cells) are essential cells in the immune system that suppress immune responses of other cells, designed to limit excessive reactions and prevent autoimmunity. Tregs are characterized by the expression of CD4, CD25, and FOXP3, while lacking CD127. CD4+FOXP3+ regulatory T cells have been referred to as "naturally-occurring" regulatory T cells to distinguish them from "suppressor" T cell populations that are generated in vitro. While other variants of suppressive T cells do exist, such as CD8 suppressor cells, Th3 and Tr-1 cells, Tregs are classically defined as CD4+CD25+FOXP3+ cells. Here, we provide a protocol for immunofluorescent staining of FOXP3 in frozen sections, adapted from a protocol from Dr. Matthias Hardtke-Wolenski, Medizinische Hochschule Hannover. This protocol has been successfully used on mouse spleen, liver, and skin sections.

Proper citation: Sam Li 2019. Immunofluorescent Staining of Foxp3 in Frozen Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.97ch9iw Copy   

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Authors: Sam Li
Group: BioLegend
Summary: BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.Note: Due to the properties of our beads, it may be possible to use far fewer beads that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.

Proper citation: Sam Li 2019. MojoSort™ Human anti-PE Nanobeads Column Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7aghibw Copy   

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