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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 2017. Flex-T™ HLA Class I ELISA Protocol. protocols.io dx.doi.org/10.17504/protocols.io.mcsc2we Copy
Authors: Kelsey Miller
Summary: Note: If the percentage of CD45+ cells in your sample is less than 50%, please follow Protocol A. If it is higher than 50% then please follow protocol B.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.
Proper citation: Kelsey Miller 2016. MojoSort™ Human CD45 Nanobeads Protocol B. protocols.io dx.doi.org/10.17504/protocols.io.e22bgge Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: Cell Fixation and Permeabilization Protocol Using 70% Ethanol
Proper citation: Kelsey Miller 2016. Ethanol Fixation Protocol. protocols.io dx.doi.org/10.17504/protocols.io.e24bggw 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: 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: 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: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2016. Immunohistochemistry Protocol for Sternberger Monoclonal Antibodies. protocols.io dx.doi.org/10.17504/protocols.io.e2jbgcn Copy
Authors: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2016. Immunohistochemistry Protocol for Ultra Streptavidin Detection Kits (USA). protocols.io dx.doi.org/10.17504/protocols.io.fq9bmz6 Copy
Authors: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2017. Immunofluorescence Microscopy Protocol with Methanol Fixed Cells. protocols.io dx.doi.org/10.17504/protocols.io.hv9b696 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 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: Kelsey Miller, Sam Li 2019. MojoSort™ Streptavidin Nanobeads Column Protocol - Negative Selection. protocols.io dx.doi.org/10.17504/protocols.io.693hh8n Copy
Authors: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2018. Immunohistochemistry Protocol for Keratin Antibodies. protocols.io dx.doi.org/10.17504/protocols.io.tkhekt6 Copy
Authors: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2016. Propidium Iodide Cell Cycle Staining Protocol. protocols.io dx.doi.org/10.17504/protocols.io.e2mbgc6 Copy
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 dx.doi.org/10.17504/protocols.io.hvpb65n Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: Anti-BrdU Staining Protocol Using 70% Ethanol and 2N HCl
Proper citation: Kelsey Miller 2017. Anti-BrdU Staining Using 70% Ethanol and 2N HCl. protocols.io dx.doi.org/10.17504/protocols.io.hvmb646 Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: Anti-Neu5Gc Antibody Kit Protocols for Flow Cytometry, ELISA, and Western blot.
Proper citation: Kelsey Miller 2016. Anti-Neu5Gc Antibody Kit Protocols. protocols.io dx.doi.org/10.17504/protocols.io.e25bgg6 Copy
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 2016. Anti-Neu5Gc Antibody Kit Protocol - Western blot. protocols.io dx.doi.org/10.17504/protocols.io.e28bghw Copy
Authors: Kelsey Miller
Group: BioLegend
Proper citation: Kelsey Miller 2016. Intracellular Staining With True-Phos™ Perm Buffer in Cell Suspensions. protocols.io dx.doi.org/10.17504/protocols.io.e2fbgbn Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: This procedure is optimized for the isolation of 107 to 1 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.Product description and procedure summary:Non Neutrophil cells are depleted by incubating the sample with the biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads. The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells are collected. These are the 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™ Mouse Neutrophil Isolation Kit Protocol. protocols.io dx.doi.org/10.17504/protocols.io.fxnbpme Copy
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