Immunodetection with streptavidin-acid phosphatase complex on Western blots

Brower, Mark S.; Brakel, Christine L.; Garry, Kimberly E.

doi:10.1016/0003-2697(85)90286-6

Cited by 23 publications

(1 citation statement)

References 14 publications

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“…While DexDesign is intended for construction of novel de-novo D-peptides, the capability to generate a DPR similar to the D-form empirical structure should validate our redesign protocol. We selected a crystal structure of the D-amino acid containing peptide GyGlanvdessG in complex with streptavidin (PDB ID 5n8j) 33 . Streptavidin is a homotetrameric protein that binds the vitamin biotin with high affinity 34 , and is therefore commonly used in Western blotting and immunoassays 35 . A monomer of streptavidin forms a β barrel, with ligands oriented towards the interior of the barrel.…”

Section: The K* Algorithmmentioning

confidence: 99%

DexDesign: A new OSPREY-based algorithm for designingde novoD-peptide inhibitors

Guerin,

Childs,

Zhou

et al. 2024

Preprint

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With over 270 unique occurrences in the human genome, peptide-recognizing PDZ domains play a central role in modulating polarization, signaling, and trafficking pathways. Mutations in PDZ domains lead to diseases such as cancer and cystic fibrosis, making PDZ domains attractive targets for therapeutic intervention. D-peptide inhibitors offer unique advantages as therapeutics, including increased metabolic stability and low immunogenicity. Here, we introduce DexDesign, a novel OSPREY-based algorithm for computationally designing de novo D-peptide inhibitors. DexDesign leverages three novel techniques that are broadly applicable to computational protein design: the Minimum Flexible Set, K*-based Mutational Scan, and Inverse Alanine Scan, which enable exponential reductions in the size of the peptide sequence search space. We apply these techniques and DexDesign to generate novel D-peptide inhibitors of two biomedically important PDZ domain targets: CAL and MAST2. We introduce a new framework for analyzing de novo peptides-evaluation along a replication/restitution axis-and apply it to the DexDesign-generated D-peptides. Notably, the peptides we generated are predicted to bind their targets tighter than their targets' endogenous ligands, validating the peptides' potential as lead therapeutic candidates. We provide an implementation of DexDesign in the free and open source computational protein design software OSPREY.

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Section: The K* Algorithmmentioning

confidence: 99%

DexDesign: A new OSPREY-based algorithm for designingde novoD-peptide inhibitors

Guerin,

Childs,

Zhou

et al. 2024

Preprint

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Protein Blotting: A Manual

Gershoni

1988

Methods of Biochemical Analysis

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Immunoblotting techniques with picogram sensitivity in cerebrospinal fluid protein detection

et al. 1989

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Agarose isoelectric focusing followed by blotting with nitrocellulose, nylon or polyvinylidene difluoride membranes, and immunochemical detection of cerebrospinal fluid IgG with various combinations of antisera, was evaluated. Polyvinylidene difluoride proved to be an easy-to-handle and reliable membrane for protein blotting. Among immunochemical visualization reactions, the most sensitive employed biotinylated goat anti-human IgG followed by streptavidin colloidal gold conjugate and silver enhancement in 20% w/v urea, allowing a sensitivity of less then 1 picogram IgG/band.

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Immunodetection with streptavidin-acid phosphatase complex on Western blots

Cited by 23 publications

References 14 publications

DexDesign: A new OSPREY-based algorithm for designingde novoD-peptide inhibitors

DexDesign: A new OSPREY-based algorithm for designingde novoD-peptide inhibitors

Protein Blotting: A Manual

Immunoblotting techniques with picogram sensitivity in cerebrospinal fluid protein detection

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