MALP-2, TLR6 and TLR2 ligand

MALP-2, TLR6 and TLR2 ligand [NBP2-26219] - 293 cells were transfected with pCMV/TLR2-6 plasmid and pNF-kB/SEAP plasmid using Lipofectamin 2000. After 48 hrs of transfection, 50 and 100 ng/ml of MALP2 as added. Cells were incubated at 37C for 24 hrs. Transfected cell supernatant was collected and analyzed using the NF-kB SEAPorter Assay kit.

MALP-2, TLR6 and TLR2 ligand [NBP2-26219] - Evaluation of the MALP2 ligand activity on TLR2/NF-kB SEAPorter HEK 293 cell line.Cell line is a stably co-transfected cell line that expresses full-length human Toll-like receptor 2 (TLR2) and the secreted alkaline phosphatase (SEAP) reporter gene under the transcriptional control of an NF-kB response element. Cells were plated in 96-well plates at 5 x 10^4 cells/well. After 16 h, cells were stimulated with various amounts of MALP2 for 24 h. SEAP was analyzed using the SEAPorter Assay Kit. Data Summary: MALP2 specifically activated the TLR2/6-depedent NF-kB/SEAP reporter cells in a dose dependent manner (Note: HEK 293 cells endogenously express TLR6).

• Reactivity: Hu, Mu, Av
• Applications: Func, In vitro
• Concentration: Please see the protocols for proper use of this product. If no protocol is available, contact technical services for assistance.

MALP-2, TLR6 and TLR2 ligand Summary

• Immunogen: MALP-2, TLR6 and TLR2 ligand
• Specificity: MALP-2 was originally isolated from Mycoplasma fermentans. This MALP-2 corresponds to the originally isolated isomer, which expresses potent endotoxin-like activity and approaches in certain experimental systems the toxicity of LPS. For description of the stereochemistry of MALP-2 please refer to M. Morr, et al. Eur. J. Immunol. 32, 3337 (2002). The importance of the stereochemistry of the central carbon atom of the diacylglycerol group has been described in the K.M. Omueti, et al. paper (2005), see below. Formula: C99H167N19O30S
• Details of Functionality: Specific Activity is approx 2x10^8units/mg. One unit is defined as the dilution giving half maximal release of nitric oxide from C3H/HeJ mouse peritoneal exudate cells in the standard assay.

Applications/Dilutions

• Application Notes: Source: Synthetic
  Formula: C99H167N19O30S
  MW: 2135.2 Use in In- vitro assay and functional reported in scientific literature (PMID 25466255)

Reactivity Notes

Human reactivity reported in scientific literature (PMID: 24771854).Mouse reactivity reported in scientific literature (PMID: 25466255). Avian reactivity reported in scientific literature (PMID: 25967535)

Packaging, Storage & Formulations

• Storage: Store at -20C. Avoid freeze-thaw cycles.
• Buffer: 2 ug in 20 ul of sterile PBS solution containing 2.5% (v/v) 2-propanol, 25 mM n-octyl-b-D-glucopyranoside and 1% (wt/v) human serum albumin.
• Concentration: Please see the protocols for proper use of this product. If no protocol is available, contact technical services for assistance.

Notes

Disclaimer Note -Disclaimer Note - Human Serum- No test method can provide total assurance that the hepatitis B virus, hepatitis C virus, human immunodeficiency virus, or any other infectious agents are absent. Thus, all blood products, including purified proteins derived from human blood sources, should be handled at Biosafety Level 2 as recommended by the CDC\NIH manual entitled Biosafety in Microbiological and Biomedical Laboratories for potentially infectious human serum, blood specimens or proteins derived from same. Source material for the human blood product supplied to your facility has been tested for the detection of HIV antibody, Hepatitis B surface antigen, antibody to Hepatitis C, HIV 1 antigen(s), antibody to HTLV - I/II, and syphilis by FDA guidelines. All units were found to be non-reactive/negative for these tests. All human blood source material is collected in FDA licensed centers and is tested with FDA approved test kits. Human Serum- No test method can provide total assurance that the hepatitis B virus, hepatitis C virus, human immunodeficiency virus, or any other infectious agents are absent. Thus, all blood products, including purified proteins derived from human blood sources, should be handled at Biosafety Level 2 as recommended by the CDC\NIH manual entitled Biosafety in Microbiological and Biomedical Laboratories for potentially infectious human serum, blood specimens or proteins derived from same. Source material for the human blood product supplied to your facility has been tested for the detection of HIV antibody, Hepatitis B surface antigen, antibody to Hepatitis C, HIV 1 antigen(s), antibody to HTLV - I/II, and syphilis by FDA guidelines. All units were found to be non-reactive/negative for these tests. All human blood source material is collected in FDA licensed centers and is tested with FDA approved test kits.

Alternate Names for MALP-2, TLR6 and TLR2 ligand

• Macrophage-activating lipopeptide-2
• MALP 2
• MALP-2, TLR6 & TLR2 ligand
• S-[2,3-bis(Palmityloxy)-(2R)-propyl-cysteinyl-GNNDESNISFKEK]
• TLR activator MALP2

Background

MALP-2, TLR6 and TLR2 ligand sticks avidly to glass or plastic. Since it is active at very low concentrations, i.e. at high dilutions, these low amounts of material tend to become adsorbed to pipette tips and plastic or glass containers. To avoid this, prepare several dilution steps of the MALP-2, TLR6 and TLR2 ligand stock solution with medium containing 5% autologous serum or buffers with 2% HSA. The presence of 50mM octyl glucoside in the first dilution step is often beneficial and gives rise to higher activity. Avoid small volumes in large vessels. Do not filter.Because of the ester-bound fatty acids, MALP-2, TLR6 and TLR2 ligand is sensitive to alkali or acid. The medium should be preconditioned in the CO2 incubator in order to ensure that the pH does not become alkaline. Add cells in preconditioned medium as soon as possible and incubate.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Support products are guaranteed for 6 months from date of receipt.

Publications for MALP-2, TLR6 and TLR2 ligand (NBP2-26219)(13)

Publications using NBP2-26219

• Bi J, Koivisto L, Pang A et al. Suppression of alphavB6 Integrin Expression by Polymicrobial Oral Biofilms in Gingival Epithelial Cells Sci Rep 2017 Jun 30 [PMID: 28667248] (In vitro, Human)
• Battin C, Hennig A, Mayrhofer P et al. A human monocytic NF-kB fluorescent reporter cell line for detection of microbial contaminants in biological samples. PLoS ONE May 25 2017 [PMID: 28542462] (Flow, Human)
• Yanai S, Tokuhara D, Tachibana D et al. Diabetic pregnancy activates the innate immune response through TLR5 or TLR1/2 on neonatal monocyte J. Reprod. Immunol. 2016 Jun 22 [PMID: 27351455] (In vitro, Human)
• Sigola LB, Fuentes AL, Millis LM et al. Effects of toll-Like receptor ligands on RAW 264.7 macrophage morphology and zymosan phagocytosis. Tissue and Cell. 2016 Apr 22 [PMID: 27157550] (Func, Mouse)
• Heidegger S, Jarosch A, Schmickl M et al. Mycoplasma hyorhinis-Contaminated Cell Lines Activate Primary Innate Immune Cells via a Protease-Sensitive Factor. PLoS ONE. 2015 Nov 14 [PMID: 26565413] (In vitro, Func, Mouse)
  
  Details:
  MALP-2 (TLR6 and TLR2 ligand) was used at 500 ng/ml concentration for functional assays involving murine B16-F10 (B16) melanoma cell line.
• Chen J, Ng MM, Chu JJ. Activation of TLR2 and TLR6 by Dengue NS1 Protein and Its Implications in the Immunopathogenesis of Dengue Virus Infection. PLoS Pathog. 2015 Jul 01 [PMID: 26226614] (Func, Human)
  
  Details:
  MALP-2, TLR6 and TLR2 ligand was used at 50 ng/ml concentration (alongwith mock or Dengue Virus DV2-infection and LPS ) for treatments of human PBMCs, HEK293 and wild-type or TLR6-/- murine peritoneal macrophages under various experimental conditions.
• Ghonime MG, Mitra S, Eldomany RA et al. Inflammasome priming is similar for francisella species that differentially induce inflammasome activation. PLoS ONE. 2015 May 21 [PMID: 25993107] (Func, Human)
• Yong Y, Liu S, Hua G et al. Identification and functional characterization of Toll-like receptor 2-1 in geese. BMC Vet. Res. 2015 May 30 [PMID: 25967535] (Func, In vitro, Avian)
  
  Details:
  MALP-2, TLR6 and TLR2 ligand (a synthetic dipalmitoylated lipopeptide) was used at 10 ng/ml concentration in RPMI 1640 medium for 30 minutes stimulation of cultured peripheral blood mononuclear cells/PBMCs which were harvested from Magang geese.
• Godefroy E, Gallois A, Idoyaga J et al. Activation of Toll-like Receptor-2 by Endogenous Matrix Metalloproteinase-2 Modulates Dendritic-Cell-Mediated Inflammatory Responses. Cell Rep. 2014 Dec 11 [PMID: 25466255] (In-vitro, Func, Mouse)
• Koziel J, Bryzek D, Sroka A et al. Citrullination Alters Immunomodulatory Function of LL-37 Essential for Prevention of Endotoxin-Induced Sepsis. J. Immunol. 2014 Jun 01 [PMID: 24771854] (LA, Human)
  
  Details:
  Human monocyte-derived macrophages stimulated with 10 ng/ml of MALP-2, Fig 7a.
• Kusagaya H, Fujisawa T, Yamanaka K et al. Toll-like receptor-mediated airway IL-17C enhances epithelial host defense in an autocrine/paracrine manner. Am. J. Respir. Cell Mol. Biol. 2014 Jan 1 [PMID: 23944933] (In vitro, Human)
  
  Details:
  TLR ligand treatment (primary normal human bronchial epithelial cells), Figs 1, S1. CpG-ODN was used at 10 ug/ml.
• Kempaiah P, Davidson LB, Perkins DJ, Byrd TF. Cystic fibrosis CFBE41o- cells contain TLR1 SNP I602S and fail to respond to Mycobacterium abscessus. J Cyst Fibros. 2013 Feb 8 [PMID: 23403223]
• Gillaux C, Mehats C, Vaiman D et al. Functional screening of TLRs in human amniotic epithelial cells. J Immunol. 2011 Sep 1 [PMID: 21775685]