Technical support training cell signaling
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The document discusses technical training on signal transduction and CST products. It describes kinases and their role in phosphorylating effectors, and the use of phosphospecific antibodies, siRNA kits, and kinases/kinase assays. Application of phosphospecific and total antibodies are summarized for techniques like western blotting, immunohistochemistry, immunofluorescence, flow cytometry, immunoprecipitation, in cell western, and ELISA. Protocols are provided for some of these techniques.
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Dot elis asat1
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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.
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Immunofluorescence Antibody Validation Report for Anti-Caspase 9 Antibody (ST...St John's Laboratory Ltd
This antibody validation report describes the validation of an anti-Caspase 9 monoclonal antibody in immunofluorescence applications on human appendix and colon tissues. The report provides the antibody information, imaging descriptions, and immunofluorescence protocol which involves tissue processing, antigen retrieval, blocking, primary and secondary antibody incubations, counterstaining, mounting and visualization under a fluorescence microscope.ELISA Types & Procedure
The document describes four main types of ELISA assays: direct, indirect, sandwich, and competitive. It provides details on the procedure for each type. Direct ELISA uses one antibody directly conjugated to an enzyme for detection. Indirect ELISA uses an unlabeled primary antibody detected by a labeled secondary antibody, allowing for signal amplification. Sandwich ELISA requires two antibodies that bind to different epitopes of the target antigen. Competitive ELISA measures competition between a labeled antigen and unlabeled antigen in a sample.
ELISA types & procedure
ELISAs can be performed with a number of modifications to the basic procedure: direct, indirect, sandwich or competitive.
Immunofluorescence Antibody Validation Report for Anti-Raf-1 Antibody (STJ95345)
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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).
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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.
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Immunofluorescence Antibody Validation Report for Anti-CK16 Antibody (STJ96958)St John's Laboratory Ltd
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
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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-validationImmunocytochemistry Protocol
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
Immunofluorescence Antibody Validation Report for Anti-p21 Antibody (STJ94862)
Immunofluorescence Antibody Validation Report for Anti-p21 Antibody (STJ94862)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
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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
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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
- 7. 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
- 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
- 12. 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
- 13. In Cell Western / ELISA In Cell Western ELISA
- 14. 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)