Technical support training cell signaling

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Technical training CST

Michiel van der Sanden

Signaaltransductie

Effector1

Kinase

Phosphospecific antibody
Phosphorylering

Effector 3 P Effector 3
Inactief Actief y
ibod
nt
Dephosphorylering la
ta
To
Phosphatase EFFECT

CST Produkten

• Kinases and Kinase Assays

• SiRNA kits

• Antibodies

SiRNA kit

X
Kinase

Phosphospecific antibody
Phosphorylering

Substrate P Substraat
Inactief actief y
ibod
ant
t al
To

Toepassingen phosphospecifieke
en totaal antibodies
• Western Blot (WB)
• Immunohistochemistry (IHC)
• Immunofluorescentie (IF)
• Flow cytometry (F)
• Immunoprecipitatie (IP)
• In Cell Western (ICW)
• ELISA

Western blot
Classical HRP WB LICOR Fluorescent WB

- + SP600125 JNK inhibitor

p-ATF

Actin

Western blot protocol
• Cell/Tissue sample preparation in buffer
• SDS PAGE Gel
• Transfer of proteins from Gel to Blot
• Block blot with Blocking buffer for 1 h
• Incubate blot overnight in BSA/Milk TBS-T
• Wash blot 3 X
• Incubate secondary antibody 1 h
• Do ECL and exposure to blot or use
LICOR system

Immunohistochemistry/
Immunofluorescence
Immunohistochemistry Immunofluorescence

Immunohistochemistry/
Immunofluorescence
Immunohistochemistry Immunofluorescence

IHC/IF blot protocol
• Fixation of samples
• Deparaffinization of slides (paraffin sections)
• Antigen unmasking (paraffin section)
• Washing of samples
• Blocking of samples
• Incubation primary antibody overnight
• Washing of samples
• Incubation secondary antibody 1 hour
• Washing
• ABC/DAB staining (IHC)
• Mount slides (fixing them on object glas)
• Analyze them under microscope

Flow cytometry

Grote Voordeel Flow: Single cell measurement + quantificatie

Flow blot protocol
• Collection of samples
• Fixation of cells
• Permeabilization of cells
• Aliquot and incubate cells in incubation buffer
• Incubation primary antibody 30-60 minutes
• Washing of samples
• Incubation secondary antibody 30 minutes (if
necessary)
• Washing
• Analyze cells on flow cytometric unit

In Cell Western / ELISA
In Cell Western ELISA

In Cell Western / ELISA
• Coat plate with capture antibody (ELISA)
• Incubate cells on plate (ICW)/grow cells (ELISA)
• Fix cells (ICW)/ Add cell lysate to plate (ELISA)
• Wash plates
• Incubate cells with detection antibody
• Wash plates
• Add Fluorescent antibody (ICW)/ Add colour substrate
(ELISA)
• Analyze on LICOR (ICW)/ Microtiter Plate Reader
(ELISA)

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The enzyme-linked immunosorbent assay (ELISA) uses antibodies and a solid-phase enzyme immunoassay to detect the presence of a specific antigen in a liquid or wet sample. Antigens from the sample are immobilised on a solid support either non-specifically or specifically. A specific antibody is then applied over the surface so it can bind to the antigen. This antibody is linked to an enzyme or can itself be detected by an enzyme-linked secondary antibody. In the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a visible signal, most commonly a colour change in the substrate, which indicates the quantity of antigen in the sample. When the presence of an antigen is analysed, the name "direct ELISA" refers to an ELISA in which only a labelled primary antibody is used, whereas the term "indirect ELISA" refers to an ELISA in which the antigen is bound by the primary antibody which then is detected by a labelled secondary antibody.

Indirect ELISA Protocol | St John's Laboratory

Indirect ELISA Protocol | St John's Laboratory

Indirect ELISA Protocol | St John's Laboratory

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Involved in induction of the epithelial to mesenchymal transition (EMT), formation and maintenance of embryonic mesoderm, growth arrest, survival and cell migration. Binds to 3 E-boxes of the E-cadherin/CDH1 gene promoter and to the promoters of CLDN7 and KRT8 and, in association with histone demethylase KDM1A which it recruits to the promoters, causes a decrease in dimethylated H3K4 levels and represses transcription. Associates with EGR1 and SP1 to mediate tetradecanoyl phorbol acetate (TPA)-induced up-regulation of CDKN2B, possibly by binding to the CDKN2B promoter region 5'-TCACA-3. In addition, may also activate the CDKN2B promoter by itself. Anti-SNAI 1 -http://www.stjohnslabs.com/snai-1-antibody Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

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Key regulator of entry into cell division that acts as a tumor suppressor. Promotes G0-G1 transition when phosphorylated by CDK3/cyclin-C. Acts as a transcription repressor of E2F1 target genes. The underphosphorylated, active form of RB1 interacts with E2F1 and represses its transcription activity, leading to cell cycle arrest. Directly involved in heterochromatin formation by maintaining overall chromatin structure and, in particular, that of constitutive heterochromatin by stabilizing histone methylation. Recruits and targets histone methyltransferases SUV39H1, KMT5B and KMT5C, leading to epigenetic transcriptional repression. Anti-Rb -http://www.stjohnslabs.com/rb-antibody-p-94135 Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

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Transmembrane serine/threonine kinase forming with the TGF-beta type I serine/threonine kinase receptor, TGFBR1, the non-promiscuous receptor for the TGF-beta cytokines TGFB1, TGFB2 and TGFB3. Transduces the TGFB1, TGFB2 and TGFB3 signal from the cell surface to the cytoplasm and is thus regulating a plethora of physiological and pathological processes including cell cycle arrest in epithelial and hematopoietic cells, control of mesenchymal cell proliferation and differentiation, wound healing, extracellular matrix production, immunosuppression and carcinogenesis. Anti-TGFβ RII -http://www.stjohnslabs.com/tgfb-rii-antibody-p-94590 Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

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Growth factor that plays an essential role in the regulation of embryonic development, cell proliferation, cell migration, survival and chemotaxis. Potent mitogen for cells of mesenchymal origin . Required for normal proliferation and recruitment of pericytes and vascular smooth muscle cells in the central nervous system, skin, lung, heart and placenta. Required for normal blood vessel development, and for normal development of kidney glomeruli. Plays an important role in wound healing. Signaling is modulated by the formation of heterodimers with PDGFA. Anti-PDGF-B -http://www.stjohnslabs.com/pdgf-b-antibody Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

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Flow cytometry is a method used for cell counting, cell sorting, biomarker detection and protein engineering. It uses lasers to enable simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second. Cells must be suspended in a stream of fluid and incubated with fluorescent-labelled antibodies which detect the expression of cell surface and intracellular molecules. The suspension is then passed by an electronic detection apparatus. The protocol for this technique is similar to but differs from that used for indirect flow cytometry, aka fluorescent-activated cell sorting (FACS).

Direct Flow Cytometry Protocol

Direct Flow Cytometry Protocol

Direct Flow Cytometry Protocol

St John's Laboratory Ltd

Indirect flow cytometry, also known as fluorescence-activated cell sorting (FACS) is a specialised type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. It is a useful scientific instrument as it provides fast, objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest. In contrast to direct flow cytometry, FACS requires two incubation steps. Firstly with the appropriate primary antibody, then with a fluorochrome-labelled secondary antibody.

Indirect Flow Cytometry Protocol

Indirect Flow Cytometry Protocol

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Transcriptional regulator which can act both as a coactivator and a corepressor and is the critical downstream regulatory target in the Hippo signaling pathway that plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis . The core of this pathway is composed of a kinase cascade wherein STK3/MST2 and STK4/MST1, in complex with its regulatory protein SAV1, phosphorylates and activates LATS1/2 in complex with its regulatory protein MOB1, which in turn phosphorylates and inactivates YAP1 oncoprotein and WWTR1/TAZ . Plays a key role in tissue tension and 3D tissue shape by regulating cortical actomyosin network formation. Anti-YAP -http://www.stjohnslabs.com/yap-antibody-p-94815 Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

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Involved in the activation cascade of caspases responsible for apoptosis execution. Binding of caspase-9 to Apaf-1 leads to activation of the protease which then cleaves and activates caspase-3. Promotes DNA damage-induced apoptosis in a ABL1/c-Abl-dependent manner. Proteolytically cleaves poly(ADP-ribose) polymerase (PARP). Isoform 2 lacks activity is an dominant-negative inhibitor of caspase-9. Strict requirement for an Asp residue at position P1 and with a marked preference for His at position P2. It has a preferred cleavage sequence of Leu-Gly-His-Asp-|-Xaa. Inhibited by the effector protein NleF that is produced by pathogenic E.coli; this inhibits apoptosis. Anti-Caspase 9 -http://www.stjohnslabs.com/caspase-9-antibody-p-98614 Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

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Boster Biological Technology

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Serine/threonine-protein kinase that acts as a regulatory link between the membrane-associated Ras GTPases and the MAPK/ERK cascade, and this critical regulatory link functions as a switch determining cell fate decisions including proliferation, differentiation, apoptosis, survival and oncogenic transformation. RAF1 activation initiates a mitogen-activated protein kinase (MAPK) cascade that comprises a sequential phosphorylation of the dual-specific MAPK kinases (MAP2K1/MEK1 and MAP2K2/MEK2) and the extracellular signal-regulated kinases (MAPK3/ERK1 and MAPK1/ERK2). Anti-Raf-1 -http://www.stjohnslabs.com/raf-1-antibody-p-94100 Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

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PHD Finger Protein 19 is coded for by the PHF19 gene (also known as hPCL3). The Polycomb Group (PcG) is a key binding site within the protein that targets trimethylated Histone H3 at Lys-36, where it recruits the PRC2 complex. It is thought to be involved in the transition of embryonic stems cells from an active to repressed state, and research has indicated its overexpression in many types of cancers.

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Drug efflux transporter present in a number of stem cells that acts as a regulator of cellular differentiation. Able to mediate efflux from cells of the rhodamine dye and of the therapeutic drug doxorubicin. Specifically present in limbal stem cells, where it plays a key role in corneal development and repair. Anti-ABCB5 -http://www.stjohnslabs.com/abcb5-antibody-p-98611 Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

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Keratin 16 is a protein that in humans is encoded by the KRT16 gene. Keratin 16 is a type I cytokeratin. It is paired with keratin 6 in a number of epithelial tissues, including nail bed, esophagus, tongue, and hair follicles. Mutations in the gene encoding this protein are associated with the genetic skin disorders pachyonychia congenita, non-epidermolytic palmoplantar keratoderma and unilateral palmoplantar verrucous nevus. Anti-CK16 -http://www.stjohnslabs.com/ck16-antibody-p-98593 Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

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The steroid hormones and their receptors are involved in the regulation of eukaryotic gene expression and affect cellular proliferation and differentiation in target tissues. Progesterone receptor isoform B (PRB) is involved activation of c-SRC/MAPK signaling on hormone stimulation. / Isoform A: inactive in stimulating c-Src/MAPK signaling on hormone stimulation. / Isoform 4: Increases mitochondrial membrane potential and cellular respiration upon stimulation by progesterone. Anti-PR -http://www.stjohnslabs.com/pr-antibody-p-99016 Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

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This method is used to visualise the localisation and quantity of a protein of interest. The target protein is bound to by a specific primary antibody, which in turn is detected by a secondary antibody conjugated to a fluorophore. A fluorescent or confocal microscope is used to visualise the protein. Immunocytochemistry (ICC) differs from immunohistochemistry (IHC) in that the former is performed on samples of intact cells that have had most, if not all, of their surrounding extracellular matrix removed. In contrast, immunohistochemical samples are sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissue. These differences cause the samples to be prepared differently. For ICC, the sample requires permeabilisation so that the antibodies can reach the intracellular targets. Depending on the thickness of the sample, IHC samples do not require this. Do you have a technical question? Get in touch: info@stjohnslabs.com

Immunocytochemistry Protocol

Immunocytochemistry Protocol

Immunocytochemistry Protocol

St John's Laboratory Ltd

May be the important intermediate by which p53/TP53 mediates its role as an inhibitor of cellular proliferation in response to DNA damage. Binds to and inhibits cyclin-dependent kinase activity, preventing phosphorylation of critical cyclin-dependent kinase substrates and blocking cell cycle progression. Functions in the nuclear localization and assembly of cyclin D-CDK4 complex and promotes its kinase activity towards RB1. At higher stoichiometric ratios, inhibits the kinase activity of the cyclin D-CDK4 complex. Anti-p21 -http://www.stjohnslabs.com/p21-antibody Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

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Transcriptional regulator which can act both as a coactivator and a corepressor and is the critical downstream regulatory target in the Hippo signaling pathway that plays a pivotal role in organ size control and tumor suppression by restricting proliferation and promoting apoptosis . The core of this pathway is composed of a kinase cascade wherein STK3/MST2 and STK4/MST1, in complex with its regulatory protein SAV1, phosphorylates and activates LATS1/2 in complex with its regulatory protein MOB1, which in turn phosphorylates and inactivates YAP1 oncoprotein and WWTR1/TAZ . Plays a key role in tissue tension and 3D tissue shape by regulating cortical actomyosin network formation. Acts via ARHGAP18, a Rho GTPase activating protein that suppresses F-actin polymerization . Plays a key role to control cell proliferation in response to cell contact. Phosphorylation of YAP1 by LATS1/2 inhibits its translocation into the nucleus to regulate cellular genes important for cell proliferation, cell death, and cell migration . The presence of TEAD transcription factors are required for it to stimulate gene expression, cell growth, anchorage-independent growth, and epithelial mesenchymal transition (EMT) induction . / Isoform 2: Isoform 2 and isoform 3 can activate the C-terminal fragment (CTF) of ERBB4 (isoform 3). Anti-YAP-http://www.stjohnslabs.com/yap-antibody-p-94815 Join our Antibody Validation Project - http://www.stjohnslabs.com/services/antibody-validation

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Intrigued by why some of the world's largest companies (Netflix, Google, Cisco, Twitter, Uber etc) are using gRPC? In this demo based talk we delve into the world of gRPC in .Net, what it does and why we should use it. We compare the interface with both Rest and graphQL. We will show you how to implement grpc server-side in .net and in the web. Finally, I will show you how the tooling helps you deliver powerful interfaces and interact with them quickly and simply.

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Discover the top Symfony development companies that excel in creating robust and scalable web applications. Our latest blog highlights the best firms specializing in Symfony, known for their expertise in delivering high-performance solutions. Whether you’re looking to start a new project or enhance an existing one, these companies offer the skills and experience needed to bring your vision to life. Read more to find your perfect Symfony development partner.

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Technical support training cell signaling

1. Technical training CST Michiel van der Sanden

2. Signaaltransductie Effector1 Kinase Phosphospecific antibody Phosphorylering Effector 3 P Effector 3 Inactief Actief y ibod nt Dephosphorylering la ta To Phosphatase EFFECT

3. CST Produkten • Kinases and Kinase Assays • SiRNA kits • Antibodies

4. SiRNA kit X Kinase Phosphospecific antibody Phosphorylering Substrate P Substraat Inactief actief y ibod ant t al To

5. Toepassingen phosphospecifieke en totaal antibodies • Western Blot (WB) • Immunohistochemistry (IHC) • Immunofluorescentie (IF) • Flow cytometry (F) • Immunoprecipitatie (IP) • In Cell Western (ICW) • ELISA

6. Western blot Classical HRP WB LICOR Fluorescent WB - + SP600125 JNK inhibitor p-ATF Actin

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8. Immunohistochemistry/ Immunofluorescence Immunohistochemistry Immunofluorescence

9. Immunohistochemistry/ Immunofluorescence Immunohistochemistry Immunofluorescence

10. IHC/IF blot protocol • Fixation of samples • Deparaffinization of slides (paraffin sections) • Antigen unmasking (paraffin section) • Washing of samples • Blocking of samples • Incubation primary antibody overnight • Washing of samples • Incubation secondary antibody 1 hour • Washing • ABC/DAB staining (IHC) • Mount slides (fixing them on object glas) • Analyze them under microscope

11. Flow cytometry Grote Voordeel Flow: Single cell measurement + quantificatie

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13. In Cell Western / ELISA In Cell Western ELISA

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