Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly-horseradish peroxidase (PolyHRP)

Manish Mishra, Shuchita Tiwari, Anita Gunaseelan, Dongyang Li, Bruce D. Hammock, Aldrin V Gomes

Research output: Contribution to journalArticle

Abstract

Immunoassays such as ELISAs and Western blotting have been the common choice for protein validation studies for the past several decades. Technical advancements and modifications are continuously being developed to enhance the detection sensitivity of these procedures. Among them, Streptavidin-containing poly-horseradish peroxidase (PolyHRP) based detection strategies have been shown to improve signals in ELISA. The use of commercially available Streptavidin and antibodies conjugated with many HRPs (PolyHRPs) to potentially enhance the detection sensitivity in Western blotting has not been previously investigated in a comprehensive manner. The use of PolyHRP-secondary antibody instead of HRP-secondary antibody increased the Western blotting sensitivity up to 85% depending on the primary antibody used. The use of a biotinylated secondary antibody and commercially available Streptavidin-conjugated with HRP or PolyHRP all resulted in increased sensitivity with respect to antigen detection. Utilizing a biotinylated secondary antibody and Streptavidin-conjugated PolyHRP resulted in as much as a 110-fold increase in Western blotting sensitivity over traditional Western blotting methods. Quantification of troponin I in rat heart lysates showed that the traditional Western blotting method only detected troponin I in ≥2 μg of lysate while Streptavidin-conjugated PolyHRP20 detected troponin I in ≥50 ng of lysate. A modified blocking procedure is also described that eliminated the interference caused by the endogenous biotinylated proteins. These results suggest that Streptavidin-conjugated PolyHRP and PolyHRP secondary antibodies are likely to be commonly utilized for Western blots in the future.

Original languageEnglish (US)
JournalElectrophoresis
DOIs
StatePublished - Jan 1 2019

Fingerprint

Streptavidin
Horseradish Peroxidase
Western Blotting
Antibodies
Troponin I
Enzyme-Linked Immunosorbent Assay
Validation Studies
Immunoassay
Rats
Proteins
Antigens

Keywords

  • Chemiluminescence
  • Horseradish peroxidase
  • PolyHRP
  • SDS PAGE
  • Streptavidin–Biotin
  • Western blotting

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

Cite this

Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly-horseradish peroxidase (PolyHRP). / Mishra, Manish; Tiwari, Shuchita; Gunaseelan, Anita; Li, Dongyang; Hammock, Bruce D.; Gomes, Aldrin V.

In: Electrophoresis, 01.01.2019.

Research output: Contribution to journalArticle

Mishra, Manish ; Tiwari, Shuchita ; Gunaseelan, Anita ; Li, Dongyang ; Hammock, Bruce D. ; Gomes, Aldrin V. / Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly-horseradish peroxidase (PolyHRP). In: Electrophoresis. 2019.
@article{ac6fc5b75bdf48978f9d4fefe11f8eee,
title = "Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly-horseradish peroxidase (PolyHRP)",
abstract = "Immunoassays such as ELISAs and Western blotting have been the common choice for protein validation studies for the past several decades. Technical advancements and modifications are continuously being developed to enhance the detection sensitivity of these procedures. Among them, Streptavidin-containing poly-horseradish peroxidase (PolyHRP) based detection strategies have been shown to improve signals in ELISA. The use of commercially available Streptavidin and antibodies conjugated with many HRPs (PolyHRPs) to potentially enhance the detection sensitivity in Western blotting has not been previously investigated in a comprehensive manner. The use of PolyHRP-secondary antibody instead of HRP-secondary antibody increased the Western blotting sensitivity up to 85{\%} depending on the primary antibody used. The use of a biotinylated secondary antibody and commercially available Streptavidin-conjugated with HRP or PolyHRP all resulted in increased sensitivity with respect to antigen detection. Utilizing a biotinylated secondary antibody and Streptavidin-conjugated PolyHRP resulted in as much as a 110-fold increase in Western blotting sensitivity over traditional Western blotting methods. Quantification of troponin I in rat heart lysates showed that the traditional Western blotting method only detected troponin I in ≥2 μg of lysate while Streptavidin-conjugated PolyHRP20 detected troponin I in ≥50 ng of lysate. A modified blocking procedure is also described that eliminated the interference caused by the endogenous biotinylated proteins. These results suggest that Streptavidin-conjugated PolyHRP and PolyHRP secondary antibodies are likely to be commonly utilized for Western blots in the future.",
keywords = "Chemiluminescence, Horseradish peroxidase, PolyHRP, SDS PAGE, Streptavidin–Biotin, Western blotting",
author = "Manish Mishra and Shuchita Tiwari and Anita Gunaseelan and Dongyang Li and Hammock, {Bruce D.} and Gomes, {Aldrin V}",
year = "2019",
month = "1",
day = "1",
doi = "10.1002/elps.201900059",
language = "English (US)",
journal = "Electrophoresis",
issn = "0173-0835",
publisher = "Wiley-VCH Verlag",

}

TY - JOUR

T1 - Improving the sensitivity of traditional Western blotting via Streptavidin containing Poly-horseradish peroxidase (PolyHRP)

AU - Mishra, Manish

AU - Tiwari, Shuchita

AU - Gunaseelan, Anita

AU - Li, Dongyang

AU - Hammock, Bruce D.

AU - Gomes, Aldrin V

PY - 2019/1/1

Y1 - 2019/1/1

N2 - Immunoassays such as ELISAs and Western blotting have been the common choice for protein validation studies for the past several decades. Technical advancements and modifications are continuously being developed to enhance the detection sensitivity of these procedures. Among them, Streptavidin-containing poly-horseradish peroxidase (PolyHRP) based detection strategies have been shown to improve signals in ELISA. The use of commercially available Streptavidin and antibodies conjugated with many HRPs (PolyHRPs) to potentially enhance the detection sensitivity in Western blotting has not been previously investigated in a comprehensive manner. The use of PolyHRP-secondary antibody instead of HRP-secondary antibody increased the Western blotting sensitivity up to 85% depending on the primary antibody used. The use of a biotinylated secondary antibody and commercially available Streptavidin-conjugated with HRP or PolyHRP all resulted in increased sensitivity with respect to antigen detection. Utilizing a biotinylated secondary antibody and Streptavidin-conjugated PolyHRP resulted in as much as a 110-fold increase in Western blotting sensitivity over traditional Western blotting methods. Quantification of troponin I in rat heart lysates showed that the traditional Western blotting method only detected troponin I in ≥2 μg of lysate while Streptavidin-conjugated PolyHRP20 detected troponin I in ≥50 ng of lysate. A modified blocking procedure is also described that eliminated the interference caused by the endogenous biotinylated proteins. These results suggest that Streptavidin-conjugated PolyHRP and PolyHRP secondary antibodies are likely to be commonly utilized for Western blots in the future.

AB - Immunoassays such as ELISAs and Western blotting have been the common choice for protein validation studies for the past several decades. Technical advancements and modifications are continuously being developed to enhance the detection sensitivity of these procedures. Among them, Streptavidin-containing poly-horseradish peroxidase (PolyHRP) based detection strategies have been shown to improve signals in ELISA. The use of commercially available Streptavidin and antibodies conjugated with many HRPs (PolyHRPs) to potentially enhance the detection sensitivity in Western blotting has not been previously investigated in a comprehensive manner. The use of PolyHRP-secondary antibody instead of HRP-secondary antibody increased the Western blotting sensitivity up to 85% depending on the primary antibody used. The use of a biotinylated secondary antibody and commercially available Streptavidin-conjugated with HRP or PolyHRP all resulted in increased sensitivity with respect to antigen detection. Utilizing a biotinylated secondary antibody and Streptavidin-conjugated PolyHRP resulted in as much as a 110-fold increase in Western blotting sensitivity over traditional Western blotting methods. Quantification of troponin I in rat heart lysates showed that the traditional Western blotting method only detected troponin I in ≥2 μg of lysate while Streptavidin-conjugated PolyHRP20 detected troponin I in ≥50 ng of lysate. A modified blocking procedure is also described that eliminated the interference caused by the endogenous biotinylated proteins. These results suggest that Streptavidin-conjugated PolyHRP and PolyHRP secondary antibodies are likely to be commonly utilized for Western blots in the future.

KW - Chemiluminescence

KW - Horseradish peroxidase

KW - PolyHRP

KW - SDS PAGE

KW - Streptavidin–Biotin

KW - Western blotting

UR - http://www.scopus.com/inward/record.url?scp=85065676315&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85065676315&partnerID=8YFLogxK

U2 - 10.1002/elps.201900059

DO - 10.1002/elps.201900059

M3 - Article

C2 - 31021001

AN - SCOPUS:85065676315

JO - Electrophoresis

JF - Electrophoresis

SN - 0173-0835

ER -