LPS-biotin (50 μg/ml) was coupled to streptavidin-coated beads (Amersham Biosciences, GE Healthcare, Chicago, IL, USA). After extensive washing, the beads were incubated with recombinant GrA(-SA) or GrK for 1 h at RT or overnight at 4 °C by head over head rotation. Bound protein was eluted from the beads with 2x concentrated Laemmli buffer, and analyzed by SDS-PAGE followed by Instant Blue total protein staining.
Streptavidin coated beads
Manufactured by Cytiva
Sourced in Sweden
Streptavidin-coated beads are a type of lab equipment used in various biotechnological applications. Streptavidin, a protein derived from the bacterium Streptomyces, is covalently coupled to the surface of the beads. The core function of these beads is to provide a high-affinity binding platform for molecules containing biotin, a small organic compound.
Automatically generated - may contain errors
6 protocols using streptavidin coated beads
1
Granzyme Protein Binding Assay
2
Integrated HPV DNA Methylation Analysis
3
Methylation Analysis of HBx Gene
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Hidden Markov models have been successfully used to partition genomes into segments of comparable stochastic structure (Durbin et al. 1998 ). Using these models for sequence analysis performed on the CpG plugin of bioinformatics software Geneious 5.5.8 (Biomatters), CpG islands were searched in the HBV genome (Kearse et al. 2012 (link)). Bisulfite PCR was performed using an EpiTect Bisulfite Kit (Qiagen) according to the manufacturer’s protocol. One microliter of bisulfite-treated DNA was used as a template. The primers used for amplifying CpG sequences in the HBx gene are described in Supplemental Table 1. After PCR, the biotinylated strand was captured on streptavidin-coated beads (Amersham Bioscience) and incubated with sequencing primers (Supplemental Table 1). The pyrosequencing reactions were performed using the PyroMark Q24 and/or PyroMark Q24 advanced (Qiagen). Pyrosequencing quantitatively measures the methylation status of several CpG sites in a given sequence. These adjacent sites usually show highly concordant methylation. Therefore, the mean percentage of methylation at detected sites was used as a representative value for each sequence.
4
Quantitative Pyrosequencing Protocol
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The primers were designed using PyrosequencingTM Assay Design Software Version 1.0; where one of the primers was biotin-labeled. Primer sequences were as follows: Sense primer biotin-5′-GGAA GGTGTTGGAGGATATT-3′, antisense primer 5′-CCT CTCAACCCTACATATCTCTTATCA-3′, sequencing primer 5′-ATCTCTTATCAACTATCTTTCA-3′. The Platinum PCR SurperMix High Fidelity (Invitrogen, Carlsbad, CA) was used to prepare the PCR reaction solution. After the biotinylated strand was captured on streptavidin-coated beads (Amersham Bioscience, Uppsala, Sweden) and incubated with sequencing primers, the specific PCR products were then subjected to quantitative pyrosequencing analysis using a Pyrosequencing™ PyroMark MD system following the protocol provided by the manufacturer. The sequencing results were analyzed using the PyroMark Q96 software (Qiagen).
5
Bisulfite Conversion and Pyrosequencing of DNA
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Bisulfite treatment of genomic DNA was performed using the EpiTect bisulfite kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Pyrosequencing was carried out for 8 of the SRAMs, reflecting both promoter or first exon CpGi’s (CBLC, MST1R, LAD1, ESRP1, HOXB4) and non-CpGi genes (PRSS8, RAB25, AXL) (Additional file 16 : Table S9). One microliter of bisulfite-treated DNA was used for each polymerase chain reaction (PCR). After an initial hot-start at 95°C for 5 minutes, all PCR reactions ran at 95°C for 30 seconds, annealing at various temperatures for 30 seconds, and underwent an extension step at 72°C for 30 seconds. All reactions were carried out with a nested PCR step during which a biotinylated universal primer was added. After PCR, the biotinylated strand was captured on streptavidin-coated beads (Amersham Bioscience, Uppsala, Sweden) and incubated with sequencing primers. Pyrosequencing was performed with PSQ HS 96 Gold reagents on a PS QHS 96 pyrosequencer (Biotage, Uppsala, Sweden) as published previously [48 (link)].
6
Quantitative DNA Methylation Analysis
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Bisulfite PCR reaction was performed using an EpiTect Bisulfite Kit (Qiagen,
Valencia, CA) according to the manufacturer’s protocol.19 (link) One microliter of bisulfite-treated DNA was
used as a template. The primers used were 5′-AGTAATGATAATGGAAGG
GGTTA-3′ as a sense primer and
5′-TACRACTCCRAAAACTCCATA-3′ as an antisense
primer. After PCR, the biotinylated strand was captured on streptavidin-coated
beads (Amersham Bioscience, Uppsala, Sweden) and incubated with sequencing primer
(5′-TYGTTYGGTGAGGTTAGGAT-3′). Pyrosequencing
quantitatively measures the methylation status of several CpG sites in a given
promoter. These adjacent sites usually show highly concordant methylation.
Therefore, the mean percentage of methylation at detected sites was used as a
representative value for gene promoter.
Valencia, CA) according to the manufacturer’s protocol.19 (link) One microliter of bisulfite-treated DNA was
used as a template. The primers used were 5′-AGTAATGATAATGGAAGG
GGTTA-3′ as a sense primer and
5′-TACRACTCCRAAAACTCCATA-3′ as an antisense
primer. After PCR, the biotinylated strand was captured on streptavidin-coated
beads (Amersham Bioscience, Uppsala, Sweden) and incubated with sequencing primer
(5′-TYGTTYGGTGAGGTTAGGAT-3′). Pyrosequencing
quantitatively measures the methylation status of several CpG sites in a given
promoter. These adjacent sites usually show highly concordant methylation.
Therefore, the mean percentage of methylation at detected sites was used as a
representative value for gene promoter.
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