EGCG-N3 for the click chemistry was synthesized from EGCG and 6-azide-6-deoxyl-idose. The manuscript involving the detail of it's synthetic procedure is in preparation (Tanaka, H. et al., submitted for publication). HSA (1.0 mg/ml) was incubated with 1 mM EGCG-N3 in PBS (pH 7.4) at 37°C. Click chemistry was performed using the reaction mixtures containing 1.0 mg/ml protein with 1 mM CuSO4, 1 mM ascorbic acid, 0.1 mM tris((1-benzyl-1H-1,2,3- triazol-4-yl)methyl)amine (Anaspec, Inc., San Jose), and 20 μM alkyne-PEG4-biotin (Click Chemistry Tools). After incubation in the dark for 2 h at room temperature, the protein samples were boiled in the Laemmli sample buffer for 5 min, and the biotinylated proteins were then subjected to SDS-PAGE/Western blot followed by detection with HRP-conjugated NeutrAvidin and ECL.
Biotin peg4 alkyne
Manufactured by Vector Laboratories
Biotin-PEG4-Alkyne is a bifunctional reagent that consists of a biotin moiety linked to an alkyne group through a polyethylene glycol (PEG4) spacer. It can be used for the covalent labeling or conjugation of biomolecules, such as proteins, peptides, or nucleic acids, that contain azido or alkyne functional groups.
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Lab products found in correlation
Biotin alkyne, by Vector Laboratories (2 mentions)
Thpta, by Merck Group (2 mentions)
Trypsin resuspension buffer, by Promega (1 mentions)
Ammonium bicarbonate abc, by Merck Group (1 mentions)
Anti rabbit igg hrp, by Beyotime (1 mentions)
Streptavidin conjugated agarose beads, by Thermo Fisher Scientific (1 mentions)
Anti mbp, by Proteintech (1 mentions)
Anti mouse igg hrp, by Beyotime (1 mentions)
Formic acid, by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
Iodoacetamide iaa, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
To alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Evos fl auto 2 imaging system, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Azidohomoalanine, by Vector Laboratories (1 mentions)
7 protocols using biotin peg4 alkyne
1
EGCG-N3 Click Chemistry Assay
2
Click Chemistry Reagents for Bioconjugation
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DADPS biotin alkyne (catalogue no. 1331), 2‐[4‐{(bis[(1‐tert‐butyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amino)methyl}‐1H‐1,2,3‐triazol‐1‐yl]acetic acid (BTTAA) (catalogue no. 1236), alkyne‐PEG4‐biotin (catalogue no. TA105) and alkyne‐Cy5 (catalogue no. TA116) were purchased from Click Chemistry Tools, GalNAz (catalogue no. 869186‐83‐4) from Jinan Samuel Pharmaceutical Co., Ltd, streptavidin‐conjugated agarose beads (catalogue no. 20350) from Thermo Fisher. Dithiothreitol (DTT) (catalogue no. 43815), ammonium bicarbonate (ABC) (catalogue no. 09830), iodoacetamide (IAA) (catalogue no. I1149), urea (catalogue no. U5128) and formic acid (FA; catalogue no. 06473) were purchased from Sigma Aldrich, and sequencing‐grade modified trypsin (catalogue no. V5111) and trypsin resuspension buffer (catalogue no. V542A) were purchased from Promega. All organic solvents were analytical grade or better. Antibodies included anti‐HA (CST, #2367, 1 : 3000), anti‐His (MBL, D291‐3, 1 : 10 000), anti‐MBP, (Proteintech, 15 089‐1‐AP, 1 : 10 000), anti‐streptavidin‐HRP (Beyotime, A0303, 1 : 5000), anti‐mouse IgG‐HRP (CST, #7076, 1 : 10 000) and anti‐rabbit IgG‐HRP (CST, #7074, 1 : 10 000).
3
Bioorthogonal Labeling of Lung ECM
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Following antigen unmasking, the lung tissue sections were reacted to Alkyne-PEG4-biotin (Click Chemistry Tools, Cat. No. TA105) under the copper-catalyzed cycloaddition (CuAAC) condition (34) (link). All samples were blocked with 1% bovine serum albumin (BSA) in PBS for 20 min and incubated with the primary antibody for Laminin (Abcam, Cat. No. ab11575, RRID:AB_298179) at the dilution of 1:500. The slides were then stained with streptavidin conjugated to Alexa Fluor-647 (Thermo Fisher Scientific, Cat. No. S21374, RRID:AB_2336066) and Donkey-anti-Rabbit IgG conjugated to Alexa Fluor-488 (Thermo Fisher Scientific, Cat. No. A32790, RRID:AB_2762833) at the dilution of 1:500. All slides were mounted with DAPI-containing Fluoromount solution (SouthernBiotech, Cat. No. 0100-20). Single-field images were taken using ZEISS LSM 700 microscopy (RRID:SCR_017377), and stitched images were taken by EVOS FL Auto 2 Imaging System (Thermo Fisher Scientific, Waltham, MA). For quantification, three random fields were taken in each lung section. Azide→biotin intensities for each field taken were measured by Fiji software (RRID:SCR_002285) and were normalized by the Laminin coverage areas. The experimenter was blinded to the subjects' treatment. The specificity of primary antibodies was validated in our previous studies (24, (link)25) (link).
4
Enrichment and Identification of O-GlcNAcylated Proteins
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For enrichment and identification of the O-GlcNAcylated proteins, experiments were performed based on the procedure of Woo and co-workers49 . Briefly, the proteins in PBS containing 1% SDS were diluted with PBS and incubated with 100 μM THPTA (Sigma-Aldrich, 762342), 0.5 mM CuSO4, 2.5 mM fresh sodium ascorbate and 200 μM either Biotin-PEG4-Alkyne (Click Chemistry Tools, TA105) for immunoblotting or Biotin-Alkyne probe for proteomics at 37 °C for 4 h.
For biotin-immunoprecipitation, proteins were precipitated and resuspended into 100 μL PBS containing 1% SDS. The protein solutions were diluted with PBS to lower the final concentration of SDS into 0.2% and incubated with pre-washed 40 μL streptavidin beads slurry at room temperature for 2 h with gentle rotation. Beads were washed sequentially with 0.2 % SDS/PBS three times and PBS three times. Enriched proteins were eluted with SDS sample buffer and subjected to SDS-PAGE.
For biotin-immunoprecipitation, proteins were precipitated and resuspended into 100 μL PBS containing 1% SDS. The protein solutions were diluted with PBS to lower the final concentration of SDS into 0.2% and incubated with pre-washed 40 μL streptavidin beads slurry at room temperature for 2 h with gentle rotation. Beads were washed sequentially with 0.2 % SDS/PBS three times and PBS three times. Enriched proteins were eluted with SDS sample buffer and subjected to SDS-PAGE.
5
Assessing Trastuzumab-PE24 Inhibition of Protein Synthesis
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Inhibition of protein synthesis of cells by trastuzumab-PE24 was evaluated by the BONCAT method [51 (link)]. Her2-positive BT-474 cells and MDA-MB-231 Her2-negative cells were seeded (5 × 104) with RPMI 1640 medium (HyClone) containing 10% FBS (HyClone) and 1% penicillin/streptomycin (HyClone) in 6-well plates and incubated at 37 °C for 24 h in an atmosphere of 5% CO2. Trastuzumab, PE24, or trastuzumab-PE24 (0.1 nM) was added to the wells and incubated at 37 °C for 20 h. Azidohomoalanine (Click Chemistry Tools) (4 mM) was added to each well and incubated at 37 °C for 2 h. The cells were washed with chilled PBS including 1 mM MgCl2 and 0.1 mM CaCl2. The washed cells were trypsinized and harvested. The harvested cells were lysed with 1% (w/v) SDS, and the cell lysate was reacted with biotin-PEG4-alkyne (Click Chemistry Tools) by the Cu(I)-catalyzed click reaction. The biotinylated proteins were detected by western blotting using the streptavidin-HRP conjugate.
6
Enrichment and Identification of O-GlcNAcylated Proteins
7
Click Chemistry for Biotin Incorporation
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For MS analysis, 10 mg of each biological replicate plus 10 mg for the internal standard (Day0) were used, except in the experiments described in Fig. 4 E,F where 5 mg were used for each biological replicate plus 5 mg for the internal standard (Day0). For immunoblot analysis (Fig. 3 D), 4 mg of starting material was used. Sodium dodecyl sulfate was added to the homogenized tissues at final concentration of 0.5%. The homogenate was then sonicated with a tip sonicator and was divided into 0.5 mg aliquots. A click reaction was performed on each aliquot. The click reaction protocol has been previously published66 (link). In brief, for each click reaction, the following reagents were added in this order: (1) 30 μL of 1.7 mM TBTA, (2) 8 μL of 50 mM copper sulfate, (3) 8 μL of 5 mM light biotin-alkyne (C16H24N4O3S, Seterah, Eugene, OR) or heavy biotin-alkyne (C13H24N3O3S13C315N, Seterah, Eugene, OR), and (4) 8 μL of 50 mM TCEP. For the reaction described in Fig. 3 D, biotin-PEG4-alkyne from Click Chemistry Tools (Scottsdale, AZ) was used. PBS was then added to a final volume of 400 μL and the reaction was incubated for 1 h at room temperature. The click reactions for each sample were combined and precipitation was performed with 25% TCA.
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