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Biotin-PEG4-tyramide

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Physical properties
Molecular weight610.77
SolventDMSO
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501
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OverviewpdfSDSpdfProtocol


Molecular weight
610.77
Biotin-PEG4-tyramide is a reagent that has been used for tyramide signal amplification (TSA) via catalyzed reporter deposition (CARD). In CARD, a reporter enzyme, such as horseradish peroxidase (HRP) conjugated to a secondary antibody, is bound to the target of interest and catalyzes the covalent deposition of biotinyl tyramide to the sample. The sample is then probed by a detector, such as streptavidin-HRP, allowing detection via chromogenic or fluorescent methods. Biotin-PEG4-tyramide has been used in immunohistochemistry, ELISA, Western blot, and in situ hybridization applications. It tends to have improved signal/background than biotinyl tyramide in most of biological detections.

Example protocol


AT A GLANCE

Protocol Summary
  1. Fix/permeabilize/block cells or tissue
  2. Add primary antibody in blocking buffer
  3. Add HRP-conjugated secondary antibody
  4. Prepare Styramide™ working solution and apply in cells or tissue for 5-10 minutes at room temperature

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Tyramide™ stock solution (100X)

Add 100 µL of DMSO into the vial of Biotin-PEG4-labeled tyramide conjugate to make a 100X tyramide stock solution.

Note: Make single-use aliquots and store any unused 100X stock solution at -20 °C, protected from light. Avoid repeat freeze-thaw cycles.

Hydrogen peroxide stock solution (100X)

Add 10 µL of 3% hydrogen peroxide (not provided) to 90 µL of ddH2O.

Note: Prepare the 100X H2O2 solution fresh on the day of use.

PREPARATION OF WORKING SOLUTION

Tyramide™ working solution (1X)

Add 100 µL of the tyramide stock solution into 20 mL of a buffer of your choice containing 0.003% H2O2.

Note: For optimal performance, use Tris Buffer, pH=7.4.

Note: A 20 mL solution is good for 200 tests. The tyramide working solution should be used immediately and made fresh on the day of use. Avoid direct exposure to light. 

Secondary antibody-HRP working solution

Make appropriate concentration of secondary antibody-HRP working solution as per the manufacturer's recommendations.

SAMPLE EXPERIMENTAL PROTOCOL

This protocol is applicable for both cells and tissues staining.

Cell fixation and permeabilization
  1. Fix the cells or tissue with 3.7% formaldehyde or paraformaldehyde, in PBS at room temperature for 20 minutes.
  2. Rinse the cells or tissue with PBS twice.
  3. Permeabilize the cells with 0.1% Triton X-100 solution for 1-5 minutes at room temperature.
  4. Rinse the cells or tissue with PBS twice.
Tissue fixation, deparaffinization and rehydration

Deparaffinize and dehydrate the tissue according to the standard IHC protocols. Perform antigen retrieval with the preferred specific solution/protocol as needed. A protocol can be found at:

https://www.aatbio.com/resources/guides/paraffin-embedded-tissue-immunohistochemistry-protocol.html

Peroxidase labeling
  1. Optional: Quench endogenous peroxidase activity by incubating cell or tissue sample in peroxidase quenching solution (such as 3% hydrogen peroxide) for 10 minutes. Rinse with PBS twice at room temperature.

  2. Optional: If using HRP-conjugated streptavidin, it is advisable to block endogenous biotins by biotin blocking buffer.

  3. Block with preferred blocking solution (such as PBS with 1% BSA) for 30 minutes at 4 °C.
  4. Remove blocking solution and add primary antibody diluted in recommended antibody diluent for 60 minutes at room temperature or overnight at  4 °C.
  5. Wash with PBS three times for 5 minutes each.
  6. Apply 100 µL of secondary antibody-HRP working solution to each sample and incubate for 60 minutes at room temperature.

    Note: Incubation time and concentration can be varied depending on the signal intensity.

  7. Wash with PBS three times for 5 minutes each.
Tyramide™ labeling
  1. Prepare and apply 100 µL of tyramide working solution to each sample and incubate for 5-10 minutes at room temperature.

    Note: If you observe a non-specific signal, you can shorten the incubation time with the tyramide reagent.  You should optimize the incubation period using positive and negative control samples at various incubation time points. Or you can use a lower concentration of the tyramide reagent in the working solution.

  2. Rinse with PBS three times.
Counterstain and fluorescence imaging
  1. Counterstain the cell or tissue samples as needed. AAT provides a series of nucleus counterstain reagents as listed in Table 1. Follow the instruction provided with the reagents.
  2. Mount the coverslip using a mounting medium with anti-fading properties.

    Note: To ensure optimal results, it is recommended to use either ReadiUse™ microscope mounting solution (Cat. 20009) or FluoroQuest™ TSA/PSA Antifade Mounting Medium *Optimized for Tyramide and Styramide Imaging* (Cat. 44890) instead of Vectashield® mounting media. There are instances where Vectashield® mounting media may not be suitable for certain TSA/PSA conjugates.

  3. Use the appropriate filter set to visualize the signal from the Styramide labeling.

Table 1. Products recommended for nucleus counterstain.

Cat# Product Name Ex/Em (nm)
17548 Nuclear Blue™ DCS1 350/461
17550 Nuclear Green™ DCS1 503/526
17551 Nuclear Orange™ DCS1 528/576
17552 Nuclear Red™ DCS1 642/660

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Biotin-PEG4-tyramide to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM163.728 µL818.639 µL1.637 mL8.186 mL16.373 mL
5 mM32.746 µL163.728 µL327.456 µL1.637 mL3.275 mL
10 mM16.373 µL81.864 µL163.728 µL818.639 µL1.637 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Images


References


View all 18 references: Citation Explorer
High specificity and tight spatial restriction of self-biotinylation by DNA and RNA G-Quadruplexes complexed in vitro and in vivo with Heme.
Authors: Lat, Prince Kumar and Liu, Kun and Kumar, Dev N and Wong, Kenneth K L and Verheyen, Esther M and Sen, Dipankar
Journal: Nucleic acids research (2020)
Biotinylation and isolation of an RNA G-quadruplex based on its peroxidase-mimicking activity.
Authors: Li, Wei and Zeng, Weiwu and Chen, Yi and Wang, Fang and Wu, Fan and Weng, Xiaocheng and Zhou, Xiang
Journal: The Analyst (2019): 4472-4476
APEX Peroxidase-Catalyzed Proximity Labeling and Multiplexed Quantitative Proteomics.
Authors: Kalocsay, Marian
Journal: Methods in molecular biology (Clifton, N.J.) (2019): 41-55
Proximity labeling of cis-ligands of CD22/Siglec-2 reveals stepwise α2,6 sialic acid-dependent and -independent interactions.
Authors: Alborzian Deh Sheikh, Amin and Akatsu, Chizuru and Imamura, Akihiro and Abdu-Allah, Hajjaj H M and Takematsu, Hiromu and Ando, Hiromune and Ishida, Hideharu and Tsubata, Takeshi
Journal: Biochemical and biophysical research communications (2018): 854-859
Tyramide Signal Amplification Permits Immunohistochemical Analyses of Androgen Receptors in the Rat Prefrontal Cortex.
Authors: Low, Katelyn L and Ma, Chunqi and Soma, Kiran K
Journal: The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society (2017): 295-308
Identification of Siglec Ligands Using a Proximity Labeling Method.
Authors: Chang, Lanyi and Chen, Yi-Ju and Fan, Chan-Yo and Tang, Chin-Ju and Chen, Yi-Hsiu and Low, Penk-Yeir and Ventura, Albert and Lin, Chun-Cheng and Chen, Yu-Ju and Angata, Takashi
Journal: Journal of proteome research (2017): 3929-3941
Immunohistochemical Methods for Measuring Tissue Lymphangiogenesis.
Authors: Royston, Daniel J and Clasper, Steven and Jackson, David G
Journal: Methods in molecular biology (Clifton, N.J.) (2016): 35-48
Development of a highly sensitive immunohistochemical method to detect neurochemical molecules in formalin-fixed and paraffin-embedded tissues from autopsied human brains.
Authors: Goto, Satoshi and Morigaki, Ryoma and Okita, Shinya and Nagahiro, Shinji and Kaji, Ryuji
Journal: Frontiers in neuroanatomy (2015): 22
Development of a sensitive ELISA for quantification of three- and four-repeat tau isoforms in tauopathies.
Authors: Luk, Connie and Giovannoni, Gavin and Williams, David R and Lees, Andrew J and de Silva, Rohan
Journal: Journal of neuroscience methods (2009): 34-42
Levels of pregnancy-associated plasma protein A in patients with coronary artery disease.
Authors: Liu, Zhi-Yuan and Zhang, Jin-Ying and Sun, Tong-Wen and Zhang, Yan-Jun and Zhang, Li and Wang, Le-Xin
Journal: Clinical and investigative medicine. Medecine clinique et experimentale (2008): E85-9