Purification and quantitation of bacteriophage M13 using desalting spin columns and digital PCR

Reitinger, Stephan; Petriv, Oleh I.; Mehr, Kevin; Hansen, Carl L.; Withers, Stephen G.

doi:10.1016/j.jviromet.2012.06.021

Cited by 9 publications

(6 citation statements)

References 12 publications

(13 reference statements)

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“…HER2 protein was biotinylated using a biotin labeling kit at the ratio of 4 biotin/protein. Then biotin-HER2 was purified by the Zeba spin column . The peptide beads in the library were incubated with biotin-HER2 and streptavidin coated magnetic beads successively.…”

Section: Methodsmentioning

confidence: 99%

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Microarray Based Screening of Peptide Nano Probes for HER2 Positive Tumor

Wang

et al. 2015

Anal. Chem.

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Peptides are excellent biointerface molecules and diagnostic probes with many advantages such as good penetration, short turnover time, and low cost. We report here an efficient peptide screening strategy based on in situ single bead sequencing on a microarray. Two novel peptides YLFFVFER (H6) and KLRLEWNR (H10) specifically binding to the tumor biomarker human epidermal growth factor receptor 2 (HER2) with aKD of 10(-8) M were obtained from a 10(5) library. Conjugated to nanoparticles, both the H6 and H10 probes showed specific accumulation in HER2-positive tumor tissues in xenografted mice by in vivo imaging.

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Section: Methodsmentioning

confidence: 99%

“…Then biotin-HER2 was purified by the Zeba spin column. 42 The peptide beads in the library were incubated with biotin-HER2 and streptavidin coated magnetic beads successively. Then all the peptide beads were introduced into a Teflon tube (diameter 1 mm, flow rate 600 μL/min) with a magnet closely next to the outer wall of the tube.…”

mentioning

confidence: 99%

Microarray Based Screening of Peptide Nano Probes for HER2 Positive Tumor

Wang

et al. 2015

Anal. Chem.

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“…These results, indicate that of the most viral genome was packaged, and the difference between VGC and PFU quantification was not caused by free unpackaged genome. Reitinger et al [22] also observed tenfold difference between VGC (resistant to DNase I treatment) and PFU using absolute quantification by digital PCR. Similar results were also observed in MS2 phage quantification [23,24].…”

Section: Resultsmentioning

confidence: 94%

A qPCR Targeted Against the Viral Replication Origin Designed to Quantify Total Amount of Filamentous Phages and Phagemids

et al. 2019

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Filamentous bacteriophages are widely used in phage display technology. The most common quantification method is lysis plaque formation test (PFT). This technique has several disadvantages, and only quantifies infective phages and is not effective when phagemids are used. We developed a qPCR method directed against the M13 replication origin, which detects between 3.3 9 10 3 and 3.3 9 10 8 viral genome copies with a linearity of R 2 = 0.9998. Using this method we were able to observe a difference of approximately ten more phages than with the PFT. This difference was not due to the presence of a free genome, which suggests the presence of non-infective particles. Using a DNaseI treatment, we observed the presence of 30% to 40% of unpackaged genome in recombinant phage modified in PIII or PVIII. The qPCR method with a DNase I treatment is an efficient method to quantify the total amount of filamentous phages.

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“…As a negative control (C-) we used bacteriophage fd (1 μL, 10 8 virions), not containing a foreign insert; as a positive control (C+), recombinant protein B7-2 (Sino Biological, China; 1 μL, 2.5 μg/mL). Cellulose was incubated with a blocking Tris buffer solution (15 mL) containing 1% BSA and 0.1% polysorbate 20 to prevent nonspecific interaction [8].…”

Section: Isolation Of Single-stranded Dna Of Bacteriophages Of the Librarymentioning

confidence: 99%

Search for Peptides Specifically Binding with the B7-2 Costimulatory Molecule

et al. 2021

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Abstract— The interaction of B7-1/B7-2 ligands with CD28/CTLA-4 receptors plays a key role in the regulation of the immune response. The aim of this study was to find and study peptides that interact with the human B7-2 molecule. In the course of the work, three rounds of affinity selection were carried out and individual phage clones were selected, which include peptides with varying degrees of interaction with the coregulatory target B7-2. As a result of DNA sequencing of selected phages, nucleotide sequences encoding peptides that specifically bind to B7-2 were obtained. The identified peptides can be used as a basis for the development of immunotherapeutic drugs for regulating the immune response in the treatment of oncological diseases.

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Purification and quantitation of bacteriophage M13 using desalting spin columns and digital PCR

Cited by 9 publications

References 12 publications

Microarray Based Screening of Peptide Nano Probes for HER2 Positive Tumor

Microarray Based Screening of Peptide Nano Probes for HER2 Positive Tumor

A qPCR Targeted Against the Viral Replication Origin Designed to Quantify Total Amount of Filamentous Phages and Phagemids

Search for Peptides Specifically Binding with the B7-2 Costimulatory Molecule

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