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5 fam ser pro leu ala gln ala val arg ser ser ser arg lys 5 tamra nh2

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5-FAM-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Lys(5-TAMRA)-NH2 is a synthetic peptide consisting of the sequence Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Lys. The peptide is labeled with the fluorescent dyes 5-FAM (5-carboxyfluorescein) and 5-TAMRA (5-carboxytetramethylrhodamine) at the N-terminus and C-terminus, respectively.

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Lab products found in correlation

Fluostar optima plate reader, by BMG Labtech (2 mentions) Ab2051, by Abcam (2 mentions) Delta f96 microwell, by Thermo Fisher Scientific (2 mentions) Varioskan flash multimode reader, by Thermo Fisher Scientific (1 mentions) Methoxysuccinyl ala ala pro val 7 amido 4 methylcoumarin, by Merck Group (1 mentions) Recombinant human hmgb1 1690 hmb, by R&D Systems (1 mentions) P38 mapk, by Santa Cruz Biotechnology (1 mentions) Sc 80652, by Santa Cruz Biotechnology (1 mentions) Tlr4 sirna, by Thermo Fisher Scientific (1 mentions) Ponceau s solution, by Merck Group (1 mentions) 4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions) Control sirna, by Thermo Fisher Scientific (1 mentions) Tapi 2, by Cayman Chemical (1 mentions) Adam10 sirna, by Thermo Fisher Scientific (1 mentions) Adam17 sirna, by Thermo Fisher Scientific (1 mentions) Adam10, by Santa Cruz Biotechnology (1 mentions) Ab13556, by Abcam (1 mentions)

4 protocols using 5 fam ser pro leu ala gln ala val arg ser ser ser arg lys 5 tamra nh2

1

Measurement of ADAM17 Activity in MEFs

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Control wildtype and Pacs2−/− MEFs were lysed at 4°C in RIPA containing protease inhibitor cocktail. The lysates were cleared by centrifugation, and protein concentration equalized by BCA assay. A black Nunc Delta F96 Microwell plate was pre-coated overnight at 4°C with a 1:100 dilution of ADAM17 antibody (ab2051, Abcam). Subsequently, the plate was washed three times in PBST (PBS with 0.05% Tween 20), and blocked in 1% BSA in PBS for 1 hour at 37°C. After washing three times in PBST, 25 µg/well of lysates were added in triplicate and incubated for 2 hours at 4°C. After washing the plate again, 5 µM of ADAM17 substrate II (5-FAM-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Lys(5-TAMRA)-NH2, ENZO Life Sciences) was added to each well for analysis of ADAM17 activity. Fluorimetric measurements with excitation at 485nm and emission at 535nm were made at 37°C with an Optima FLUOSTAR plate reader (BMG Labtech), programmed for measuring every 5 minutes for 1 hour.
Dombernowsky S.L., Samsøe-Petersen J., Petersen C.H., Instrell R., Hedegaard A.M., Thomas L., Atkins K.M., Auclair S., Albrechtsen R., Mygind K.J., Fröhlich C., Howell M., Parker P., Thomas G, & Kveiborg M. (2015). The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17. Nature communications, 6, 7518.
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2

Measurement of ADAM17 Activity in MEFs

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Control wildtype and Pacs2−/− MEFs were lysed at 4°C in RIPA containing protease inhibitor cocktail. The lysates were cleared by centrifugation, and protein concentration equalized by BCA assay. A black Nunc Delta F96 Microwell plate was pre-coated overnight at 4°C with a 1:100 dilution of ADAM17 antibody (ab2051, Abcam). Subsequently, the plate was washed three times in PBST (PBS with 0.05% Tween 20), and blocked in 1% BSA in PBS for 1 hour at 37°C. After washing three times in PBST, 25 µg/well of lysates were added in triplicate and incubated for 2 hours at 4°C. After washing the plate again, 5 µM of ADAM17 substrate II (5-FAM-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Lys(5-TAMRA)-NH2, ENZO Life Sciences) was added to each well for analysis of ADAM17 activity. Fluorimetric measurements with excitation at 485nm and emission at 535nm were made at 37°C with an Optima FLUOSTAR plate reader (BMG Labtech), programmed for measuring every 5 minutes for 1 hour.
Dombernowsky S.L., Samsøe-Petersen J., Petersen C.H., Instrell R., Hedegaard A.M., Thomas L., Atkins K.M., Auclair S., Albrechtsen R., Mygind K.J., Fröhlich C., Howell M., Parker P., Thomas G, & Kveiborg M. (2015). The sorting protein PACS-2 promotes ErbB signalling by regulating recycling of the metalloproteinase ADAM17. Nature communications, 6, 7518.
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3

Enzymatic Activities of PBMC Supernatants

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PBMC supernatants enzymatic activities were analysed in 384 black wells plates in a final volume of 30 µL, using the procedure described (26 (link)). Briefly, neutrophil elastase (NE) activity was measured using the fluorogenic substrate Methoxysuccinyl-Ala-Ala-Pro-Val-7-amido-4-methylcoumarin, (Sigma), excitation and emission wavelength being 460 and 370 nm, respectively, while pan-metalloprotease activity was assessed with the substrate 5-FAM-Ser-Pro-Leu-Ala-Gln-Ala-ValArg-Ser-Ser-Ser-Arg-Lys(5-TAMRA)-NH2 (Enzo Life Sciences), excitation and emission wavelength being 485 and 535 nm, respectively. Fluorescence was read over a 3 h period, with a Varioskan™ Flash Multimode Reader (Thermo Scientific).
Gaudin C., Ghinnagow R., Lemaire F., Villeret B., Sermet-Gaudelus I, & Sallenave J.M. (2024). Abnormal functional lymphoid tolerance and enhanced myeloid exocytosis are characteristics of resting and stimulated PBMCs in cystic fibrosis patients. Frontiers in Immunology, 15, 1360716.
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4

Recombinant HMGB1 Signaling Pathway

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Recombinant human HMGB1 (1690-HMB, endotoxin less than 0.10 EU/μg) was purchased from R&D Systems (Abingdon, UK). SB203580 and dimethyl sulfoxide (DMSO) were from EMD Chemicals (Darmstadt, Germany). TAPI-2 was from Cayman Chemical (Ann Arbor, MI, USA). Antibodies to human TLR4 (76B357.1: a mouse monoclonal antibody against amino acids 100 -200 of human TLR4), RAGE (sc-80652, a mouse monoclonal antibody against a truncated extracellular domain of human RAGE), p38 mitogen-activated protein kinase (p38 MAPK), ADAM10 and actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies to human TLR4 (ab13556; a rabbit polyclonal antibody against amino acids 420-435 of human TLR4) and ADAM17 were from Abcam. An antibody to human phospho-p38 MAPK (Thr180/Tyr182) was from Cell Signaling Technology (Denvers, MA, USA).
ADAM17-siRNA, ADAM10-siRNA, RAGE-siRNA, TLR4-siRNA and control-siRNA (Ambion ® ) were from Life Technologies (Paisley, UK). The fluorogenic substrate, 5FAM-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Ser-Arg-Lys(5-TAMRA)-NH 2 , was from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). 4',6-Diamidino-2phenylindole dihydrochloride (DAPI) and Ponceau S solution were from Sigma-Aldrich (St. Louis, MO, USA).
Yang W.S., Kim J.J., Lee M.J., Lee E.K, & Park S.K. (2018). Ectodomain Shedding of RAGE and TLR4 as a Negative Feedback Regulation in High-Mobility Group Box 1-Activated Aortic Endothelial Cells. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 51(4).
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