Protein transfer from fixed, stained, and dried polyacrylamide gels and immunoblot with protein A-gold

Perides, Georg; Plagens, Ulrich; Traub, Peter

doi:10.1016/0003-2697(86)90125-9

Cited by 26 publications

(12 citation statements)

References 10 publications

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“…Gels stained with Coomassie blue R250 (UNIT 10.6) can be effectively immunoblotted by the following procedure, based on Perides et al (1986) and Dionisi et al (1995). Briefly, the stained gel is soaked in a series of solutions designed to increase the solubility of the proteins after staining and fixation.…”

Section: Blotting Of Stained Gelsmentioning

confidence: 99%

Immunoblotting and Immunodetection

Gallagher¹,

Winston²,

Fuller³

et al. 2008

CP Molecular Biology

100

102

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Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.

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Section: Blotting Of Stained Gelsmentioning

confidence: 99%

Immunoblotting and Immunodetection

Gallagher¹,

Winston²,

Fuller³

et al. 2008

CP Molecular Biology

100

102

View full text Add to dashboard Cite

show abstract

“…Gels stained with Coomassie blue R250 (UNIT 10.6; Sasse and Gallagher, 2009) can be effectively immunoblotted by the following procedure, based on Perides et al (1986) and Dionisi et al (1995). Briefly, the stained gel is soaked in a series of solutions, as described, in order to increase the solubility of the proteins after staining and fixation.…”

Section: Blotting Of Stained Gelsmentioning

confidence: 99%

Immunoblotting and Immunodetection

Gallagher¹,

Winston²,

Fuller³

et al. 2016

CP Molecular Biology

View full text Add to dashboard Cite

Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with fluorophores, horseradish peroxidase, or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate fluorescent imaging technologies or with chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed.

show abstract

“…Nitrocellulose and Polyvinylidene fluoride (PDVF) are the most popular membranes in Western blotting. Fixing or staining of gels will lower the efficiency of transfer, especially for the proteins more than 50 kDa (Perides et al, 1986). Post transfer staining might be applied by using compatible reversible stains such as Ponceau-S Red, CPTS (Copper Phthalocyanine Tetrasulfonic Acid) amido Black and Spyro Ruby (need laser to visualize) or non-compatible stains like Coomassie Brilliant Blue and Colloidal Gold (known to block some epitopes) (Millipore).…”

Section: Western Blotting (Also Known As Immunoblotting)mentioning

confidence: 99%