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Cytokines, small proteins (about 5-20 kDa), are very important in human health and disease, specifically in host responses to infection, immune responses, inflammation, trauma, sepsis, cancer, and reproduction. Similar to hormones, cytokines are important in cell signaling: released by certain types of cells, they affect the behavior of other cells and sometimes the releasing cell itself. Different from hormones, cytokines circulate in much higher concentrations and are produced by various types of cells, such as immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, endothelial cells, fibroblasts, and stromal cells. More than one type of cells may produce the same cytokine. Cytokines act through receptors to modulate the balance between humoral and cell-based immune responses. Cytokines regulate the maturation, growth, and responsiveness of particular cell populations. Some cytokines enhance or inhibit the action of other cytokines in complex ways.

Cytokines can be classed as the following based on their presumed functions, cell of secretion, or target of action:

Interleukins – produced mainly by T-helper cells.
Chemokines – mediating chemoattraction (chemotaxis) between cells.
Lymphokines – produced by lymphocytes.
Monokines – produced primarily by monocytes and macrophages.
Interferons – involved in antiviral responses.
Colony stimulating factors – supporting the growth of cells in semisolid media.

Interleukins:

Type I (grouped by receptor subunit):

γ-chain subfamily:
IL2 / IL15
IL4 / IL13
IL7
IL9
IL21

β-chain subfamily:
IL3
IL5
GM-CSF

IL6 like/gp130 subfamily:
IL6
IL11
IL27
IL30
IL31
+non IL OSM
LIF
CNTF
CTF1

IL-12/IL12RB1 subfamily:
IL12
IL23
IL27
IL35

Others:
IL14
IL16
IL32
IL34

Type II:

IL 10 subfamily:
IL10 / IL22
IL19
IL20
IL24
IL26
Interferon type III (IL28 / IFNL2+3 and IL29 / IFNL1)

Interferon I subfamily:
IFNA1
IFNA2
IFNA4
IFNA5
IFNA6
IFNA7
IFNA8
IFNA10
IFNA13
IFNA14
IFNA16
IFNA17
IFNA21
IFNB1
IFNK
IFNW1

Interferon II subfamily:
IFNG

Ig superfamily:

IL1A / IL1F1
IL1B / IL1F2
1Ra / IL1F3
IL1F5
IL1F6
IL1F7
IL1F8
IL1F9
IL1F10
IL-33 / IL1F11
IL-18 / IL1G

IL 17 family:

IL17/IL25 (IL17A)

Chemokines:

CCL subfamily:
CCL1
CCL2 / MCP-1
CCL3 / MIP-1α
CCL4 / MIP-1β
CCL5 / RANTES
CCL6
CCL7
CCL8
CCL9
CCL11
CCL12
CCL13
CCL14
CCL15
CCL16
CCL17
CCL18 / PARC / DC-CK1 / AMAC-1 / MIP-4
CCL19
CCL20
CCL21
CCL22
CCL23
CCL24
CCL25
CCL26
CCL27
CCL28

CXCL subfamily:
CXCL1/KC
CXCL2
CXCL3
CXCL4
CXCL5
CXCL6
CXCL7
CXCL8 / IL8
CXCL9
CXCL10
CXCL11
CXCL12
CXCL13
CXCL14
CXCL15
CXCL16
CXCL17

CX3CL subfamily:
CX3CL1

XCL subfamily:
XCL1
XCL2

TNF:

TNFA
Lymphotoxin
TNFB/LTA
TNFC/LTB
TNFSF4
TNFSF5/CD40LG
TNFSF6
TNFSF7
TNFSF8
TNFSF9
TNFSF10
TNFSF11
TNFSF13B
EDA

Monokines:

Interleukin 1 (IL1-alpha and IL1-beta)
TNF-alpha
TNF-beta / LTA
CSF1
GM-CSF
G-CSF

Lymphokines:

Th1 subfamily:
IFNG
TNFB

Th2 subfamily:
IL4
IL5
IL6
IL10
IL13

Others:

KITLG
Colony-stimulating factors: CSF1/M-CSF, CSF2/GM-CSF, and CSF3/G-CSF
SPP1

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Heparin, a highly sulfated glycosaminoglycan with the highest negative charge density of any known biological molecule, is located in the extracellular matrix and plays important physiological roles in anticoagulation and angiogenesis. The acidic polysaccharide of heparin consists of a heterogeneous disaccharide repeating unit of hexosamine and uronic acid (L-iduronic or D-glucuronic acid) connected through 1-4 linkages and modified with various functional groups. Conserved among a number of widely different species, heparin is usually produced by basophils and mast cells and released only into the vasculature at sites of tissue injury. In addition to its well-known usage as an injectable anticoagulant, heparin can also be used to form an inner anticoagulant surface on various experimental and medical devices such as test tubes and renal dialysis machines. Whether the right dose of heparin is being used to a patient can be monitored by the activated partial thromboplastin time (aPTT), a blood test measuring the time that it takes the blood plasma to clot.

The three heparinase enzymes (heparinase I, heparinase II, and heparinase III) produced by Flavobacterium heparinum specifically recognize and cleave at different sequences of heparin. Heparinase I enzyme (EC 4.2.2.7) cleaves heparin at the linkages between hexosamines and O-sulfated iduronic acids, yielding oligoaccharides (mainly disaccharides). In addition, Heparinase I cleaves heparin at the antithrombin III binding pentasaccharide domain. Heparinase II enzyme cleaves at the 1-4 linkages between hexosamines and uronic acid residues (both glucuronic and iduronic) of heparin, yielding oligoaccharides (mainly disaccharides). Heparinase III enzyme (EC 4.2.2.8) does not cleaves heparin.

Heparin can bind to RNA polymerase and interfere with DNA transcription by blocking RNA polymerase from binding to the promoter region. Heparin can also bind to DNA and interfere with PCR in diagnostic procedures if the sample is from the patient treated with heparin. Heparinase I and II enzymes can be used to treat the samples and eliminate the interference caused by heparin.

The activated clotting time (ACT) is the most commonly used method for monitoring heparin during cardiopulmonary bypass surgery. Heparinase I can be used to treat the specimen in ACT to monitor non-heparin-related alterations in coagulation function.

Heparin contamination in specimens is a common cause of the unexpected PTT prolongation. By treating the plasma with heparinase I, one can determine if the PTT prolongation is due to heparin.

Thromboelastography (TEG) during cardiopulmonary bypass (CPB) to diagnose excessive postoperative hemorrhage can be interfered by even small amount of heparin, producing a nondiagnostic tracing. Heparinase can be used to treat TEG blood samples and remove the interference caused by heparin.

In addition to neutralization of heparin, the single and mixture of heparinases can be used for detection and determination of plasma heparin, quality control in heparin manufacturing, and preparation of low molecular weight heparins, disaccharide or oligoaccharide from unfractionated heparin.

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The cheapest but easy and effective way to get a plasmid miniprep kit is to buy spin columns for plasmid miniprep alone and make solutions with the following buffer recipe:

Buffer P1: 50 mM Tris-HCl, 10 mM EDTA, pH 8.0 (25°C, the same below), 50 ug/ml RNase A
Buffer P2: 0.2 M NaOH, 1% SDS
Buffer N3: 4 M guanidine HCl, 0.5 M potassium acetate, pH 4.2
Buffer PB (washing): 5 M guanidine HCl, 20 mM Tris-HCl, pH 6.6, 38% ethanol
Buffer PE (washing): 100 mM NaCl, 10 mM Tris-HCl, pH 7.5, add 4 volume ethanol before use
Buffer EB (elution): 10 mM Tris-HCl, pH 8.5

The protocols of various commercial plasmid miniprep kits are similar.

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The mainstream genome editing tools include clustered regularly interspaced short palindromic repeat (CRISPR)/CAS9 RNA-guided nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and helper dependent adenoviral vectors (HDAdVs). The process and protocols of mainstream genome editing methods from literatures:

1). the CRISPR-Cas9 system:

– Genome engineering of mammalian haploid embryonic stem cells using the Cas9/RNA system.
The stem stable cell line development method uses the CRISPR system in combination with haploid embryonic stem cells (ESCs) to manipulate the mammalian genome.

– CasOT: a genome-wide Cas9/gRNA off-target searching tool.
A local tool designed to find potential off-target sites.

– Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells.
A method to influence the differentiation state of human pluripotent stem cells (hPSCs) with a CRISPR-associated catalytically inactive dCas9 fused to an effector domain.

– Genome-scale CRISPR-Cas9 knockout screening in human cells.
Lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells.

– Genetic screens in human cells using the CRISPR-Cas9 system.
A pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines.

– Genome engineering using the CRISPR-Cas9 system.
A set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. It takes 1-2 weeks to do target design and gene modifications, and 2-3 weeks to obtain modified clonal cell lines.

– CRISPR Genome Engineering Resources
Comprehensive CRISPR genome engineering resources organized by Zhang’s lab.

– Cas9 as a versatile tool for engineering biology.
Advances of the Cas9 targeting methodology, and potential applications ranging from basic science to the clinic.

2). zinc finger nucleases (ZFNs):

– Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery.

3). transcription activator-like effector nucleases (TALENs):

– Genetic engineering of human pluripotent cells using TALE nucleases.

4). helper dependent adenoviral vectors (HDAdVs):

Disclaimer:
We index the protocols for your information only. It does not necessarily mean that we agree with all of them. You rather than Syd Labs takes full responsibility for using any information described here.

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The general hybridoma antibody cloning protocol and antibody sequencing protocol seem very straightforward: RNA extraction, reverse transcription to cDNA, nested PCR amplifications, cloning of PCR products, sequencing, and analysis of sequences. However, it has taken scientists a long time to optimize a variety of parameters in the protocols, especially V-gene specific primers and antibody sequence databases. Hybridoma antibody cloning and antibody sequencing protocols from public literatures:

– Sequencing of antibodies.
Detailed protocol for antibody cloning and antibody sequencing.

– Antibody variable region sequencing as a method for hybridoma cell line authentication.
Variable region sequencing used for discrimination between hybridoma cell lines.

– Cloning, expression, and modification of antibody V regions.
Detailed protocol for cloning and expression of immunoglobulin variable regions using PCR with redundant primers.

– Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3′ to 5′ exonuclease activity.
Degenerate primers for PCR amplification of mouse VH and VL. A single highly degenerate FR1 primer sufficient for VL amplification; a series of FR1 primers for VH amplification.

– A general method allowing the design of oligonucleotide primers to amplify the variable regions from immunoglobulin cDNA.
A rational method of designing primers to amplify VH and VL for framework 1 (FR1) of immunoglobulins.

– Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family.
Two sets of oligonucleotide 5′-primers hybridizing the relatively conserved motifs within the signal sequences of the 15 heavy chain and 18 kappa light chain gene families.

– Rapid cloning of any rearranged mouse immunoglobulin variable genes.
Designing of 5′ and 3′ universal primers based on regions highly conserved across all analyzed VH and VL gene families, and the joining or constant regions.

– Primer design for the cloning of immunoglobulin heavy-chain leader-variable regions from mouse hybridoma cells using the PCR.
A set of universal primers using conserved sequences of leader (signal peptide), framework one and constant regions of the immunoglobulin heavy-chain genes.

– A general method for chimerization of monoclonal antibodies by inverse polymerase chain reaction which conserves authentic N-terminal sequences.
The method circumvents the potential problems brought by degenerate primers matching to framework region 1 and to the joining regions.

– Rapid amplification of cDNA ends (RACE) improves the PCR-based isolation of immunoglobulin variable region genes from murine and human lymphoma cells and cell lines.
A reliable and versatile RACE PCR method for the isolation of VH genes from human and murine lymphoma cells, especially if consensus primer PCR fails.

– Cloning and sequencing of human immunoglobulin V lambda gene segments.
14 (including 4 pseudogenes) new human variable (V) gene segments of lambda light chains from a single individual.

– Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction.
The first method established to use PCR to amplify and clone mouse immunoglobulin (Ig) variable (V) regions after selective first-strand cDNA synthesis.

The procedure of hybridoma antibody cloning and sequencing sounds very straightforward. However, many factors affects the efficiency and success rate. Experienced scientists at Syd Labs provides hybridoma antibody cloning and antibody sequencing services if the project is difficult for you.

Disclaimer:
We index the protocols for your information only. It does not necessarily mean that we agree with all of them. You rather than Syd Labs takes full responsibility for using any information described here.

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Similar to process of hybridoma or clonal B cell antibody cloning and sequencing, the work flow of single B cell antibody cloning and sequencing is: separation of peripheral blood mononuclear cells (PBMC) from blood, staining of PBMC with the B cell selective marker (CD19 antibody), sorting of stained B cells by flow cytometry, total RNA extraction, cDNA synthesis, two-step PCR amplification, sequencing of the PCR products, and sequence analysis with V region germline database and the Genbank database. Single B cell antibody cloning and antibody sequencing protocols from public literatures:

– Sequencing heavy- and light-chain variable genes of single B-hybridoma cells by total enzymatic amplification.
A two-step amplification protocol.

– Systematic design and testing of nested RT-PCR primers for specific amplification of mouse rearranged/expressed immunoglobulin variable region genes from small number of B cells.
112 nested primers to amplify the rearranged / expressed VH and VL genes from any mouse immunoglobulin V gene family from a single or a small number of B cells.

– Human immunoglobulin variable region gene analysis by single cell RT-PCR.
A detailed protocol for single B cell antibody cloning and antibody squencing.

– Amplification of IgG VH and VL (Fab) from single human plasma cells and B cells.
A simple FACS-based single cell RT-PCR protocol for the amplification of correctly paired IgG Fab from single human B cells or plasma cells.

The technique of single B cell antibody cloning and antibody sequencing is attractive but challenging for most academia and industrial clients. Experienced scientists at Syd Labs provide single B cell antibody cloning service and antibody sequencing service if the project is difficult for you.

Disclaimer:
We index the protocols for your information only. It does not necessarily mean that we agree with all of them. You rather than Syd Labs takes full responsibility for using any information described here.

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Two mainstream strategies have been used to sequence antibody repertoires. The classical strategy is to design primers to cover as many functional V-genes as possible and create a diverse antibody libraries. The modern strategy is to use high-throughput next generation sequencing (NGS) to “deeply” sequencing of binding populations. The abundance of antibody mRNA can also be analyzed in the same time. High-throughput antibody repertoire sequencing protocols from public literatures:

– Degenerate primer design to clone the human repertoire of immunoglobulin heavy chain variable regions.
Identification of the most highly conserved region in framework 1 and framework 4 using iCODEHOP. A set of degenerated 5′ primers for the V(H) genes from human peripheral blood mononuclear cells.

– V-gene amplification revisited – An optimised procedure for amplification of rearranged human antibody genes of different isotypes.
Near to 100% of all functional and putatively functional V-genes in VBASE2 successfully used to amplify rearranged antibody genes of different isotypes.

– MAD-DPD: designing highly degenerate primers with maximum amplification specificity.
A new set of primers for amplification of antibody variable fragments from mouse spleen cells, which theoretically covers very diverse antibody sequences.

The technique of high-throughput sequencing of the antibody repertoire is attractive but challenging for most scientists in academia and industry. Experienced scientists at Syd Labs provide high-throughput antibody repertoire sequencing services if the project is difficult for you.

Disclaimer:
We index the protocols for your information only. It does not necessarily mean that we agree with all of them. You rather than Syd Labs takes full responsibility for using any information described here.

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The key for successful scFv phage library construction is efficient two-step PCR amplification of heavy chain (VH) and ligh chain (VL) variable regions. scFv phage library construction and scFv phage library screening protocols from public literatures:

– Efficient amplification of light and heavy chain variable regions and construction of a non-immune phage scFv library.
scFv library for V(kappa) and V(H) genes.

– A compact phage display human scFv library for selection of antibodies to a wide variety of antigens.
A human single chain variable fragment (scFv) library of 1.5 x 10^8 individual clones from 140 non-immunized human donors.

– Optimal construction of non-immune scFv phage display libraries from mouse bone marrow and spleen established to select specific scFvs efficiently binding to antigen.
A non-immune library from mouse bone-marrow and spleen.

– Generation of a large complex antibody library from multiple donors.
A large complex library of IgM and IgG single chain antibodies from 50 donors.

– Cloning and Expression of Single-Chain Fragments (scFv) from Mouse and Rat Hybridomas.
A variety of primer sets for complex mouse libraries consisting of more than 10^5 different antibody sequences.

– Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.
A optimized phage display system with examples of scFvs derived from spleen-cell repertoires of mice immunized with ampicillin and from hybridoma cell lines.

The procedure of scFv phage library construction and screening sounds very straightforward. However, many factors affects the efficiency and success rate. Experienced scientists at Syd Labs provide scFv phage library construction service and scFv phage library screening service if the projects are difficult for you.

Disclaimer:
We index the protocols for your information only. It does not necessarily mean that we agree with all of them. You rather than Syd Labs takes full responsibility for using any information described here.

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The technique of antibody humanization is attractive but challenging for most scientists in academia and industry. Experienced scientists at Syd Labs provide antibody humanization service if the project is difficult for you.

Disclaimer:
We index the protocols for your information only. It does not necessarily mean that we agree with all of them. You rather than Syd Labs takes full responsibility for using any information described here.

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