After 24 hours under flow (as above), HUVECs were incubated for 30 minutes with 500 μM biotin tyramide (Iris Biotech, Marktredwitz, Germany) under flow. Cells were removed from flow and the Ibidi chamber was flushed once and basal media supplemented with 1 mM H2O2 was added for a total exposure time of 1 minute. The labelling reaction was quenched by three washes with PBS containing 10 mM sodium azide, 10 mM ascorbate, 5 mM Trolox. Cells were lysed in RIPA buffer (Sigma-Aldrich, UK), supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich, UK) and 10 mM sodium azide (to inhibit HRP activity). Biotinylated content was pulled down with PierceHigh Capacity Streptavidin Agarose (Thermo Fisher Scientific, UK). Beads were washed three times with lysis buffer before lysate was added. Beads were rotated with lysate overnight at 4°C and washed three times with either PBS supplemented with 10 mM sodium azide and Protease Inhibitor Cocktail. Biotinylated content was released from beads for immunoblotting by boiling for 5 minutes in 1x SDS-PAGE sample buffer(Thermofisher) with 5% β-mercaptoethanol.
Biotin tyramide
Manufactured by Iris Biotech
Sourced in Germany
Biotin tyramide is a reagent used in various biochemical techniques, such as immunohistochemistry and in situ hybridization. It functions as a substrate for the enzyme horseradish peroxidase (HRP), which catalyzes its deposition onto target molecules, enabling signal amplification and detection.
Automatically generated - may contain errors
Lab products found in correlation
Ls 3500, by Iris Biotech (2 mentions)
Pierce high capacity streptavidin agarose, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Agarose, by Merck Group (1 mentions)
Sybr green 2, by Thermo Fisher Scientific (1 mentions)
Ultrapure dnase rnase free distilled water, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Sodium phosphate dibasic, by Merck Group (1 mentions)
Sodium phosphate monobasic, by Merck Group (1 mentions)
Urea, by Daejung Chemicals (1 mentions)
Nigericin, by Merck Group (1 mentions)
Tlrl b5lps, by InvivoGen (1 mentions)
Monensin, by Cayman Chemical (1 mentions)
Cl097, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Merck Group (1 mentions)
Streptavidin alexa 546, by Thermo Fisher Scientific (1 mentions)
6 protocols using biotin tyramide
1
Biotinylation and Pulldown of Proteins
2
Preparation and Analysis of Biomolecular Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 1
Sodium chloride, potassium chloride, sodium phosphate monobasic, sodium phosphate dibasic, agarose, DMSO (dimethyl sulfoxide), hemin, ABTS, and H2O2 were purchased from Sigma Aldrich. UltraPure Dnase/Rnase-Free distilled water, SYBR green II, ThT, and FBS were purchased from Invitrogen. 30% acrylamide-bis-acrylamide solution (29:1), APS (ammonium persulfate), and 10×TBE (Tris/boric acid/EDTA) buffer were purchased from PanReac Applichem. Biotin tyramide was purchased from Iris Biotech, and streptavidin was purchased from Rockland Immunochemicals. Urea was purchased from Daejung.
UltraPure Dnase/Rnase-Free distilled water was used for all experiments unless otherwise specified. The annealing protocol in this disclosure was a gradual cooling from 95° C. to 4° C. at 0.5° C./30s unless otherwise specified.
3
Preparation and Storage of Immunostimulatory Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nigericin (Sigma N7143) was resuspended in ethanol (20 mM) and stored at −20 °C. LPS-B5 (Invivogen tlrl-b5lps) was resuspended in ultrapure water, aliquoted, and stored at −20 °C. Monensin (Cayman Chemicals #16488) was dissolved in ethanol (10 mM) immediately before use. CL097 (Sigma SML2566) was resuspended in ultrapure water (5 mg/mL) with dropwise addition of 35% HCl, aliquoted, and stored at −80 °C. Imiquimod hydrochloride (MedChemExpress HY-B0180A) was resuspended in ultrapure water with sonication (4 mg/mL), aliquoted, and stored at −80 °C. Biotin tyramide (Iris biotech LS-3500) was resuspended in DMSO (500 mM), aliquoted, and stored at −80 °C. PMA (Sigma P1585) was resuspended in DMSO (1 mg/mL), aliquoted, and stored at −80 °C. All other chemical reagents are listed in Table S15 .
4
Biocytin-filled Neuron Visualization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Slices with biocytin-filled neurons were fixed overnight in PFA 4% and then washed three times in PB. Subsequently, slices were left in 0.5% (vol/vol) avidin-biotinylated HRP complex (ABC) solution that contained PBS (4% NaCl)-Tx 0.5% (vol/vol) for 24 h at 4°C. The slices were washed in PB, and then incubated with tyramide signal amplification (TSA) (for details see Krabichler et al., 2017 (link)). Briefly, sections were incubated in 0.0001% biotin-tyramide (IRIS Biotech GmbH, Marktredwitz, Germany; Cat# LS-3500, Lot. 1407008) and 0.003% H2O2 in 0.05 M borate buffer, pH 8.5, for 24 h at 4°C. Finally, and after washing in PB, slices were incubated in Streptavidin-Alexa 546 (Thermo Fisher Scientific, Waltham, MA, USA) diluted 1:500 in PBS (4% NaCl)-Tx 0.5% for 24 h at 4°C.
5
Affinity Purification of APEX2-Labeled Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four 15-cm plates of confluent HUVECs were required for each proteomics condition analyzed. HUVECs were nucleofected with APEX2-eGFP-Rab27a or buffer alone and seeded into 15-cm plates. Transfected HUVECs were incubated overnight with 7 μM heme to encourage effective folding of APEX2. Mock and APEX2-eGFP-Rab27a−transfected cells were fed with 500 μM biotin tyramide (Iris Biotech) (30 minutes, 37°C). HUVECs were untreated or stimulated with 12-myristate 13-acetate (PMA) or histamine/adrenaline/3-isobutyl-1-methylxanthine (HAI). After 10 minutes, the culture medium was replaced with M199 supplemented with 1 mM hydrogen peroxide. After 1 minute, the biotinylation reaction was stopped by 3 washes in stop solution (phosphate-buffered saline [PBS], 10 mM sodium azide, 10 mM ascorbate, and 5 mM Trolox). HUVECs were lysed in RIPA buffer with 10 mM sodium azide and protease inhibitors. Lysates centrifuged (21 000g, 15 minutes, 4°C) and protein concentration determined (Pierce 660 nm, Thermo Fisher Scientific). Protein lysate from each condition was saved for confirmatory immunoblotting. A 1.8-mg quantity of lysate was added to 250 μL high-capacity neutravidin beads (Life Technologies) in low-binding tubes (Life Technologies) and incubated overnight at 4°C. The beads were washed (25 mM ammonium bicarbonate) before centrifugation and storage at −80°C.
6
Stable APEX Fusion Protein Expression
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!