SDS-PAGE and Western Blotting: Basic Principles and Protocol | Springer Nature Experiments (2025)

Table of Contents
Abstract Figures (0) & Videos (0) Experimental Specifications Techniques Reagents Other Keywords Techniques Reagents Other Keywords Related articles E6 Tagged Protein Production, Extraction, and Purification from Escherichia coli Lysate Enrichment and Detection of Wnt Proteins from Cell Culture Media A Novel Methodology for Stripping and Reprobing of Western Blots Originally Developed with Colorimetric Substrate TMB E6 Tagged Protein Production, Extraction, and Purification from Escherichia coli Lysate Enrichment and Detection of Wnt Proteins from Cell Culture Media A Novel Methodology for Stripping and Reprobing of Western Blots Originally Developed with Colorimetric Substrate TMB Phosphoprotein Detection on Protein Electroblot Using a Phosphate-Specific Fluorophore Diagnostic Assays for Myeloperoxidase and Myeloperoxidase Deficiency SDS-PAGE Immunoblot Analysis Determination of a Transcription Factor-Binding Site by Nuclease Protection Footprinting onto Southwestern Blots Phosphoprotein Detection on Protein Electroblot Using a Phosphate-Specific Fluorophore Diagnostic Assays for Myeloperoxidase Deficiency Determination of a Transcription-Factor-Binding Site by Nuclease Protection Footprinting onto Southwestern Blots References Abstract Experimental Specifications Techniques Reagents Other Keywords Techniques Reagents Other Keywords Related articles E6 Tagged Protein Production, Extraction, and Purification from Escherichia coli Lysate Enrichment and Detection of Wnt Proteins from Cell Culture Media A Novel Methodology for Stripping and Reprobing of Western Blots Originally Developed with Colorimetric Substrate TMB E6 Tagged Protein Production, Extraction, and Purification from Escherichia coli Lysate Enrichment and Detection of Wnt Proteins from Cell Culture Media A Novel Methodology for Stripping and Reprobing of Western Blots Originally Developed with Colorimetric Substrate TMB Phosphoprotein Detection on Protein Electroblot Using a Phosphate-Specific Fluorophore Diagnostic Assays for Myeloperoxidase and Myeloperoxidase Deficiency SDS-PAGE Immunoblot Analysis Determination of a Transcription Factor-Binding Site by Nuclease Protection Footprinting onto Southwestern Blots Phosphoprotein Detection on Protein Electroblot Using a Phosphate-Specific Fluorophore Diagnostic Assays for Myeloperoxidase Deficiency Determination of a Transcription-Factor-Binding Site by Nuclease Protection Footprinting onto Southwestern Blots References Figures (0) & Videos (0) References

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SDS-PAGE and Western Blotting: Basic Principles and Protocol | Springer Nature Experiments (3)

Authors:

Mukesh Bhatt 1 ,

Vishal Rai 2 ,

Ashok Kumar 2 ,

Kiran 2

... R. K. Avasthe 1

Mukesh Bhatt 1 ,

Vishal Rai 2 ,

Ashok Kumar 2 ,

Kiran 2 ,

Ajay Kumar Yadav 2 ,

Kaushal Kishor Rajak 2 ,

Vikas Gupta 3 ,

Vishal Chander 2 ,

R. K. Avasthe 1

Show more details

Series: Springer Protocols Handbooks > Book: Protocols for the Diagnosis of Pig Viral Diseases

Protocol | DOI: 10.1007/978-1-0716-2043-4_23

Affiliations:

  1. ICAR Research Complex for NEH Region, Sikkim Centre, Gangtok, Sikkim, India
  2. ICAR-IVRI, Bareilly, UP, India
  3. National Institute of Animal Health, Baghpat, UP, India

Less

Citations: 2

SDS-PAGE and Western Blotting: Basic Principles and Protocol | Springer Nature Experiments (6) Access enabled via: An Institution

Abstract

Western blotting is an important analytical technique used in cell and molecular biology for last four decades. It involves separation of proteins in SDS-PAGE and then transfer of proteins to a membrane followed by detection. By using a western blot,

…more

Western blotting is an important analytical technique used in cell and molecular biology for last four decades. It involves separation of proteins in SDS-PAGE and then transfer of proteins to a membrane followed by detection. By using a western blot, one can identify specific protein from a complex mixture of proteins. Along with its use as a diagnostic aid, it can also be used to verify proteins of interest in exploratory proteomic studies to identify different disease mechanisms. The ease of performing the technique, low cost, and accessibility further support the use of western blot in proteomic research. However, a good understanding, initial training, and optimization are of utmost importance because being a multi-step technique, it is prone to false results and incorrect interpretation. This chapter attempts to explain the technique and theory behind western blot along with some ways to troubleshoot.

less

Figures (0) & Videos (0)

Experimental Specifications

Techniques

Reagents

Other Keywords

SDS-PAGE

Western Blot

Gel Electrophoresis

Colorimetry

Electroblotting

Antibodies

Protein

Experimental Models

Kits

Please check back later as we are actively working on gathering the necessary information. Thank you for your understanding and patience.

Protein blotting

Techniques

SDS-PAGE and Western Blotting: Basic Principles and Protocol | Springer Nature Experiments (11)

SDS-PAGE

Western Blot

Gel Electrophoresis

Colorimetry

Electroblotting

Reagents

SDS-PAGE and Western Blotting: Basic Principles and Protocol | Springer Nature Experiments (12)

Please check back later as we are actively working on gathering the necessary information. Thank you for your understanding and patience.

Other Keywords

SDS-PAGE and Western Blotting: Basic Principles and Protocol | Springer Nature Experiments (13)

Protein blotting

Related articles

Based on techniques

E6 Tagged Protein Production, Extraction, and Purification from Escherichia coli Lysate

Diana Gomes et al., 2023, Springer Protocols

Enrichment and Detection of Wnt Proteins from Cell Culture Media

Pooja R. Sonavane & Karl Willert , 2022, Springer Protocols

A Novel Methodology for Stripping and Reprobing of Western Blots Originally Developed with Colorimetric Substrate TMB

Parmita Kar et al., 2015, Springer Protocols

Show more related articles

E6 Tagged Protein Production, Extraction, and Purification from Escherichia coli Lysate

Diana Gomes et al., 2023, Springer Protocols

Enrichment and Detection of Wnt Proteins from Cell Culture Media

Pooja R. Sonavane & Karl Willert , 2022, Springer Protocols

A Novel Methodology for Stripping and Reprobing of Western Blots Originally Developed with Colorimetric Substrate TMB

Parmita Kar et al., 2015, Springer Protocols

Phosphoprotein Detection on Protein Electroblot Using a Phosphate-Specific Fluorophore

Lee Broderick Bockus & R. Hal Scofield , 2015, Springer Protocols

Diagnostic Assays for Myeloperoxidase and Myeloperoxidase Deficiency

William M. Nauseef, 2014, Springer Protocols

SDS-PAGE Immunoblot Analysis

Patricia Klemmer , 2011, Springer Protocols

Determination of a Transcription Factor-Binding Site by Nuclease Protection Footprinting onto Southwestern Blots

Athanasios G. Papavassiliou , 2009, Springer Protocols

Phosphoprotein Detection on Protein Electroblot Using a Phosphate-Specific Fluorophore

Lee Broderick Bockus & R. Hal Scofield , 2009, Springer Protocols

Diagnostic Assays for Myeloperoxidase Deficiency

William M. Nauseef, 2007, Springer Protocols

Determination of a Transcription-Factor-Binding Site by Nuclease Protection Footprinting onto Southwestern Blots

Athanasios G. Papavassiliou, 2001, Springer Protocols

less

References

  1. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76(9):4350
  2. Burnette WN (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate—polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112(2):195–203
  3. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98(3):503–517

    4. Show more references

  4. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76(9):4350
  5. Burnette WN (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate—polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112(2):195–203
  6. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98(3):503–517
  7. Alwine JC, Kemp DJ, Stark GR (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A 74(12):5350–5354
  8. Gwozdz T, Dorey K (2017) Western blot. In: Basic science methods for clinical researchers. Academic, Boca Raton, pp 99–117
  9. Counts SE (2010) Western blot. In: Encyclopedia of movement disorders, vol 2010. Academic, pp 323–326 . ISBN 9780123741059.https://doi.org/10.1016/B978-0-12-374105-9.00297-5
  10. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
  11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275
  12. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150(1):76–85
  13. Kurien BT, Scofield RH (2015) Western blotting: an introduction. Methods Mol Biol 1312:17–30. . PMID: 26043986; PMCID: PMC7304528https://doi.org/10.1007/978-1-4939-2694-7_5
  14. Wilson K, Walker JM, Hofmann A, Clokie S (2018) Wilson and Walker’s principles and techniques of biochemistry and molecular biology. Cambridge University Press, New York, NY
  15. Kurien BT, Scofield RH (2009) Protein blotting and detection: methods and protocols. Humana Press, New York
  16. Kattoor JJ, Saurabh S, Malik YS, Sircar S, Dhama K, Ghosh S, Bányai K, Kobayashi N, Singh RK (2017) Unexpected detection of porcine rotavirus C strains carrying human origin VP6 gene. Vet Q 37(1):252–261.https://doi.org/10.1080/01652176.2017.1346849
  17. Todd D, McNulty MS, Allan GM (1984) The use of polyacrylamide gel electrophoresis of virus RNA in the study of rotavirus infections. In: McNulty MS, McFerran JB (eds) Recent advances in virus diagnosis. Current topics in veterinary medicine and animal science, vol 29. Springer, Dordrecht.https://doi.org/10.1007/978-94-009-6039-8_11
  18. Urzainqui A, Tabarés E, Carrasco L (1987) Proteins synthesized in African swine fever virus-infected cells analyzed by two-dimensional gel electrophoresis. Virology 160(1):286–291.https://doi.org/10.1016/0042-6822(87)90076-6
  19. Bhatt M, Mohapatra JK, Pandey LK, Mohanty NN, Das B, Prusty BR, Pattnaik B (2018) Mutational analysis of foot and mouth disease virus nonstructural polyprotein 3AB-coding region to design a negative marker virus. Virus Res 243:36–43.https://doi.org/10.1016/j.virusres.2017.10.010
  20. Greenlee JJ, Kunkle RA, Smith JD, Greenlee MHW (2016) Scrapie in swine: a diagnostic challenge. Food Saf 4(4):110–114. . PMID: 32231914; PMCID: PMC6989210https://doi.org/10.14252/foodsafetyfscj.2016019
  21. Hedman C, Otero A, Douet JY, Lacroux C, Lugan S, Filali H, Corbière F, Aron N, Badiola JJ, Andréoletti O, Bolea R (2018) Detection of PrPres in peripheral tissue in pigs with clinical disease induced by intracerebral challenge with sheep-passaged bovine spongiform encephalopathy agent. PLoS One 13(7):e0199914.https://doi.org/10.1371/journal.pone.0199914
  22. Ameri M, Zhou EM, Hsu WH (2006) Western blot immunoassay as a confirmatory test for the presence of anti-mycoplasma hyopneumoniae antibodies in swine serum. J Vet Diagn Investig 18(2):198–201.https://doi.org/10.1177/104063870601800210
  23. Plotzki E, Keller M, Ivanusic D, Denner J (2016) A new western blot assay for the detection of porcine cytomegalovirus (PCMV). J Immunol Methods 437:37–42.https://doi.org/10.1016/j.jim.2016.08.001
  24. Yang S, Li L, Yin S et al (2018) Single-domain antibodies as promising experimental tools in imaging and isolation of porcine epidemic diarrhea virus. Appl Microbiol Biotechnol 102:8931–8942.https://doi.org/10.1007/s00253-018-9324-7

    25. less

Abstract

Western blotting is an important analytical technique used in cell and molecular biology for last four decades. It involves separation of proteins in SDS-PAGE and then transfer of proteins to a membrane followed by detection. By using a western blot,

…more

Western blotting is an important analytical technique used in cell and molecular biology for last four decades. It involves separation of proteins in SDS-PAGE and then transfer of proteins to a membrane followed by detection. By using a western blot, one can identify specific protein from a complex mixture of proteins. Along with its use as a diagnostic aid, it can also be used to verify proteins of interest in exploratory proteomic studies to identify different disease mechanisms. The ease of performing the technique, low cost, and accessibility further support the use of western blot in proteomic research. However, a good understanding, initial training, and optimization are of utmost importance because being a multi-step technique, it is prone to false results and incorrect interpretation. This chapter attempts to explain the technique and theory behind western blot along with some ways to troubleshoot.

less

Experimental Specifications

Techniques

Reagents

Other Keywords

SDS-PAGE

Western Blot

Gel Electrophoresis

Colorimetry

Electroblotting

Antibodies

Protein

Experimental Models

Kits

Please check back later as we are actively working on gathering the necessary information. Thank you for your understanding and patience.

Protein blotting

Techniques

SDS-PAGE and Western Blotting: Basic Principles and Protocol | Springer Nature Experiments (20)

SDS-PAGE

Western Blot

Gel Electrophoresis

Colorimetry

Electroblotting

Reagents

SDS-PAGE and Western Blotting: Basic Principles and Protocol | Springer Nature Experiments (21)

Please check back later as we are actively working on gathering the necessary information. Thank you for your understanding and patience.

Other Keywords

SDS-PAGE and Western Blotting: Basic Principles and Protocol | Springer Nature Experiments (22)

Protein blotting

Related articles

Based on techniques

E6 Tagged Protein Production, Extraction, and Purification from Escherichia coli Lysate

Diana Gomes et al., 2023, Springer Protocols

Enrichment and Detection of Wnt Proteins from Cell Culture Media

Pooja R. Sonavane & Karl Willert , 2022, Springer Protocols

A Novel Methodology for Stripping and Reprobing of Western Blots Originally Developed with Colorimetric Substrate TMB

Parmita Kar et al., 2015, Springer Protocols

Show more related articles

E6 Tagged Protein Production, Extraction, and Purification from Escherichia coli Lysate

Diana Gomes et al., 2023, Springer Protocols

Enrichment and Detection of Wnt Proteins from Cell Culture Media

Pooja R. Sonavane & Karl Willert , 2022, Springer Protocols

A Novel Methodology for Stripping and Reprobing of Western Blots Originally Developed with Colorimetric Substrate TMB

Parmita Kar et al., 2015, Springer Protocols

Phosphoprotein Detection on Protein Electroblot Using a Phosphate-Specific Fluorophore

Lee Broderick Bockus & R. Hal Scofield , 2015, Springer Protocols

Diagnostic Assays for Myeloperoxidase and Myeloperoxidase Deficiency

William M. Nauseef, 2014, Springer Protocols

SDS-PAGE Immunoblot Analysis

Patricia Klemmer , 2011, Springer Protocols

Determination of a Transcription Factor-Binding Site by Nuclease Protection Footprinting onto Southwestern Blots

Athanasios G. Papavassiliou , 2009, Springer Protocols

Phosphoprotein Detection on Protein Electroblot Using a Phosphate-Specific Fluorophore

Lee Broderick Bockus & R. Hal Scofield , 2009, Springer Protocols

Diagnostic Assays for Myeloperoxidase Deficiency

William M. Nauseef, 2007, Springer Protocols

Determination of a Transcription-Factor-Binding Site by Nuclease Protection Footprinting onto Southwestern Blots

Athanasios G. Papavassiliou, 2001, Springer Protocols

less

Advertisement

References

  1. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76(9):4350
  2. Burnette WN (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate—polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112(2):195–203
  3. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98(3):503–517

    4. Show more references

  4. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76(9):4350
  5. Burnette WN (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate—polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112(2):195–203
  6. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98(3):503–517
  7. Alwine JC, Kemp DJ, Stark GR (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A 74(12):5350–5354
  8. Gwozdz T, Dorey K (2017) Western blot. In: Basic science methods for clinical researchers. Academic, Boca Raton, pp 99–117
  9. Counts SE (2010) Western blot. In: Encyclopedia of movement disorders, vol 2010. Academic, pp 323–326 . ISBN 9780123741059.https://doi.org/10.1016/B978-0-12-374105-9.00297-5
  10. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
  11. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275
  12. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150(1):76–85
  13. Kurien BT, Scofield RH (2015) Western blotting: an introduction. Methods Mol Biol 1312:17–30. . PMID: 26043986; PMCID: PMC7304528https://doi.org/10.1007/978-1-4939-2694-7_5
  14. Wilson K, Walker JM, Hofmann A, Clokie S (2018) Wilson and Walker’s principles and techniques of biochemistry and molecular biology. Cambridge University Press, New York, NY
  15. Kurien BT, Scofield RH (2009) Protein blotting and detection: methods and protocols. Humana Press, New York
  16. Kattoor JJ, Saurabh S, Malik YS, Sircar S, Dhama K, Ghosh S, Bányai K, Kobayashi N, Singh RK (2017) Unexpected detection of porcine rotavirus C strains carrying human origin VP6 gene. Vet Q 37(1):252–261.https://doi.org/10.1080/01652176.2017.1346849
  17. Todd D, McNulty MS, Allan GM (1984) The use of polyacrylamide gel electrophoresis of virus RNA in the study of rotavirus infections. In: McNulty MS, McFerran JB (eds) Recent advances in virus diagnosis. Current topics in veterinary medicine and animal science, vol 29. Springer, Dordrecht.https://doi.org/10.1007/978-94-009-6039-8_11
  18. Urzainqui A, Tabarés E, Carrasco L (1987) Proteins synthesized in African swine fever virus-infected cells analyzed by two-dimensional gel electrophoresis. Virology 160(1):286–291.https://doi.org/10.1016/0042-6822(87)90076-6
  19. Bhatt M, Mohapatra JK, Pandey LK, Mohanty NN, Das B, Prusty BR, Pattnaik B (2018) Mutational analysis of foot and mouth disease virus nonstructural polyprotein 3AB-coding region to design a negative marker virus. Virus Res 243:36–43.https://doi.org/10.1016/j.virusres.2017.10.010
  20. Greenlee JJ, Kunkle RA, Smith JD, Greenlee MHW (2016) Scrapie in swine: a diagnostic challenge. Food Saf 4(4):110–114. . PMID: 32231914; PMCID: PMC6989210https://doi.org/10.14252/foodsafetyfscj.2016019
  21. Hedman C, Otero A, Douet JY, Lacroux C, Lugan S, Filali H, Corbière F, Aron N, Badiola JJ, Andréoletti O, Bolea R (2018) Detection of PrPres in peripheral tissue in pigs with clinical disease induced by intracerebral challenge with sheep-passaged bovine spongiform encephalopathy agent. PLoS One 13(7):e0199914.https://doi.org/10.1371/journal.pone.0199914
  22. Ameri M, Zhou EM, Hsu WH (2006) Western blot immunoassay as a confirmatory test for the presence of anti-mycoplasma hyopneumoniae antibodies in swine serum. J Vet Diagn Investig 18(2):198–201.https://doi.org/10.1177/104063870601800210
  23. Plotzki E, Keller M, Ivanusic D, Denner J (2016) A new western blot assay for the detection of porcine cytomegalovirus (PCMV). J Immunol Methods 437:37–42.https://doi.org/10.1016/j.jim.2016.08.001
  24. Yang S, Li L, Yin S et al (2018) Single-domain antibodies as promising experimental tools in imaging and isolation of porcine epidemic diarrhea virus. Appl Microbiol Biotechnol 102:8931–8942.https://doi.org/10.1007/s00253-018-9324-7

    25. less

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  • SDS-PAGE and Western Blotting: Basic Principles and Protocol | Springer Nature Experiments (29)

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SDS-PAGE and Western Blotting: Basic Principles and Protocol | Springer Nature Experiments (2025)

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