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Authors: Sam Li
Group: BioLegend
Proper citation: Sam Li 2019. Anti-BrdU Staining Protocols Using DNAse with Surface and Fluorescent Proteins. protocols.io dx.doi.org/10.17504/protocols.io.bac9iaz6 Copy
Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary: Target cells are positively selected or depleted by incubating the sample with an anti-human APC conjugated antibody, followed by incubation with magnetic anti-APC Nanobeads. The magnetically labeled fraction is retained by the use of a magnetic separator. These are the APC+ cells, do not discard them if those are the cells of interest. Some of the 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 anti-APC Nanobeads Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7xhhpj6 Copy
Authors: Kelsey Knight
Group: BioLegend
Summary: Catalog Number: 658502
Storage Temperature: 2°C-8°C
Product 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: Kelsey Knight 2018. Anti-c-Myc Tag (9E10) Affinity Gel Protocol. protocols.io dx.doi.org/10.17504/protocols.io.tideka6 Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: The cells targeted by the Streptavidin Nanobeads are either selected or depleted by incubating your sample with the magnetic particles after incubating with a biotin-conjugated antibody or antibody cocktail. 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.
Proper citation: Kelsey Miller 2016. MojoSort™ Streptavidin Nanobeads Protocol. protocols.io dx.doi.org/10.17504/protocols.io.e2rbgd6 Copy
Authors: Kelsey Miller, 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 and less antibody 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: Kelsey Miller, Sam Li 2019. MojoSort™ Streptavidin Nanobeads Column Protocol - Positive Selection. protocols.io dx.doi.org/10.17504/protocols.io.692hh8e Copy
Authors: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2018. Th9 Polarization of Mouse CD4+ Cells. protocols.io dx.doi.org/10.17504/protocols.io.tnnemde Copy
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.
Proper citation: Sam Li 2020. MojoSort™ Mouse CD8a Selection Kit Column Protocol. protocols.io dx.doi.org/10.17504/protocols.io.bbugintw Copy
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 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 2019. MojoSort™ Human anti-APC Nanobeads Column Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7afhibn Copy
Authors: Sam Li
Group: BioLegend
Summary: Buyer is solely responsible for determining whether Buyer has all intellectual property rights that are necessary for Buyer's intended uses of the BioLegend TotalSeq™ products. For example, for any technology platform Buyer uses with TotalSeq™, it is Buyer's sole responsibility to determine whether it has all necessary third party intellectual property rights to use that platform and TotalSeq™ with that platform.
TotalSeq™-B antibodies are designed to be used with 10x Single Cell 3’ Reagent Kit v3 with Feature Barcoding. The adjusted protocol below is for customers who are utilizing TotalSeq™-A antibodies with the v3 kit instead. Note: Step 4 from the 10x Genomics user guide document number CG000185, Rev B only applies to the cDNA library and NOT to the TotalSeq™-A library.
Please read the entire protocol below and the 10x Genomics user guide for the Chromium Single Cell 3ʹ Reagent Kits v3 with Feature Barcoding technology for Cell Surface Protein before starting the experiments. 10x Genomics user guide document number CG000185, Rev B.
Commonly used abbreviations:ADT: Antibody derived tags, the oligo sequence conjugated to regular TotalSeq™-A antibodiesHTO: Hashtag oligonucleotides, the oligo sequence conjugated to TotalSeq™-A Hashtag antibodies
Proper citation: Sam Li 2019. TotalSeq™-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v3 3.1 Protocol. protocols.io dx.doi.org/10.17504/protocols.io.8aahsae Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: The HLA class I ELISA is an enzyme immunoassay based on the detection of β2-microglobulin subunit of HLA class I complexes, after capturing the complex through the conjugated biotin. To this end, biotinylated HLA class I complex is first captured in streptavidin coated microtiter wells. Subsequently, HRP-conjugated anti-human β2-microglobulin is added to detect intact HLA class I complexes. Only intact HLA class I complexes are recognized. Peptides with high affinity binding will be clearly detected by this ELISA technique, while peptides with a moderate to low binding affinity for HLA class I provide a moderate to non-detectable signal. This protocol is designed to evaluate the efficiency of peptide exchange when using the Flex-T™ system.
Proper citation: Kelsey Miller 2016. Flex-T™ HLA Class I ELISA Protocol. protocols.io dx.doi.org/10.17504/protocols.io.e7ebhje Copy
Authors: Sam Li
Group: BioLegend
Proper citation: Sam Li 2019. Sandwich ELISA Protocol. protocols.io dx.doi.org/10.17504/protocols.io.98xh9xn Copy
Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary:
This kit is designed for the sequential positive selection of CD4+ T cells from human peripheral blood mononuclear cells (PBMCs). Human monocytes express both CD14 and CD4. When using only CD4 Nanobeads for positive selection of human CD4 T cells, monocytes could be isolated along with the T cells. If this monocyte fraction does not impact your application, there is no need to address it. However, not including this population may be required. Thus, the first step in this kit is the depletion of CD14+ cells using a combination of biotin anti-human CD14 and Streptavidin Nanobeads. The second step is the positive selection of the CD4 T cells using directly conjugated CD4 Nanobeads. 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™ Human CD4 T Cell Selection Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7z8hp9w Copy
Authors: BioLegend, Inc.
Group: BioLegend
Summary: This is collection of BioLegend protocols for polarization of mouse CD4+ cells. Please make sure you refer to the correct protocol, depending on whether you are using Th1, Th2, Th17, or Treg.
Proper citation: BioLegend, Inc. 2016. Polarization of Mouse CD4+ Cells. protocols.io dx.doi.org/10.17504/protocols.io.ezsbf6e Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: Cell Fixation and Permeabilization Protocol Using 70% Ethanol
Proper citation: Kelsey Miller 2017. Cell Fixation and Permeabilization Protocol using 70% Ethanol. protocols.io dx.doi.org/10.17504/protocols.io.hv3b68n Copy
Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary:Target cells are either selected or depleted by incubating the 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. If these are the cells of interest; do not discard the liquid. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.Sample Preparation:Enzymatic digestion of mouse brain followed by myelin removal is recommended to achieve the highest purity and yield. There are several protocols published that can be applied. As a general guideline, Trypsin digestion followed by a 70/37/30% percoll gradient will increase final purity and yield.Scale up volumes if using 14 ml tubes and Magnet, and place the tube in the magnet for 10 minutes.
Proper citation: Sam Li 2019. MojoSort™ Selection Kits Protocol - 5. protocols.io dx.doi.org/10.17504/protocols.io.7z9hp96 Copy
Authors: Sam Li
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 5pmol of Neu5Gc per µg 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 Neu5Gc is directly linked to the glycoprotein of interest rather than to an accompanying sample component.
Proper citation: Sam Li 2019. Anti-Neu5Gc Antibody Kit Protocol - Western Blot. protocols.io dx.doi.org/10.17504/protocols.io.97hh9j6 Copy
Authors: Sam Li
Group: BioLegend
Proper citation: Sam Li 2019. LEGEND MAX™ Human α-Synuclein ELISA Kit Protocol. protocols.io dx.doi.org/10.17504/protocols.io.98th9wn Copy
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 and less antibody cocktail that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:2 to 1:10 for the antibody cocktail can be used. Dilutions ranging from 1:5 to 1:20 for the Streptavidin Nanobeads can be used. Please contact BioLegend Technical Service ([email protected]) if further assistance is needed.
Proper citation: Sam Li 2019. MojoSort™ Mouse NK Cell Isolation Kit Column Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7bhhij6 Copy
Authors: Kelsey Knight
Group: BioLegend
Proper citation: Kelsey Knight 2018. Immunohistochemistry Protocol for Paraffin-Embedded Sections. protocols.io dx.doi.org/10.17504/protocols.io.tkjekun Copy
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™ Mouse anti-PE Nanobeads Column Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7achiaw Copy
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