5-mer telomere oligonucleotide 5′-(TTAGGG)5-3′ (Sigma-Aldrich, Dorset, UK) was 3′-biotinylated using Pierce reagents (Thermo Fisher Scientific, York, UK) and hybridized to complementary sequence oligonucleotide. Nuclear extracts were prepared using NE-PER reagents (Thermo Fisher Scientific, York, UK). EMSAs were performed using LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific, York, UK). Reactions included 2.5% glycerol, 5 mM MgCl2, 50 ng/μL poly(dI. dC), 0.05% NP40 and 20 fmol labeled probe with or without 200-fold excess of unlabeled competitor or 2.5 μg nuclear extracts. Complexes were electrophoresed at 4 °C on 6% DNA retardation gels (Thermo Fisher Scientific, York, UK) and blotted onto nylon membrane (Roche Diagnostics Ltd., West Sussex, UK). Label detection used Chemiluminescent Nucleic Acid Detection Module kit reagents (Thermo Fisher Scientific, York, UK.
Pierce reagents
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Pierce reagents are a line of laboratory reagents and consumables designed for a variety of applications in biochemistry, cell biology, and proteomics research. They are manufactured and distributed by Thermo Fisher Scientific.
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Lab products found in correlation
2 protocols using pierce reagents
1
Quantitative Telomere-Protein Binding Assay
2
Endoxylanase Activity Quantification
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Endo-β-1,4-xylanase activity was measured by the release of reducing sugars according to the Somogyi-Nelson method (Nelson, 1944) .
One unit of endoxylanase activity was defined as the corresponding to the release of 1 μmol of reducing sugar per minute. The standard reaction mixture consisted of 2.5%
xylan, 50 mM sodium acetate (AcONa) buffer (pH 5) and the appropriate dilution of the purified enzyme or culture crude extract. Standard assays were incubated at 50 ºC and 1,200 rpm for 5 and 10 min, in order to check the linearity of the activity.
Enzymatic activity against glucose-containing substrates was measured following the release of glucose using the Glucose-TR kit (Spinreact, Vall d'en Bas, Spain) according to the manufacturer's instructions.
The BCA method was used to quantify proteins, using bovine serum albumin (BSA) as standard and Pierce reagents (Thermo scientific, Waltham, MA, USA), following the manufacturer's instructions.
One unit of endoxylanase activity was defined as the corresponding to the release of 1 μmol of reducing sugar per minute. The standard reaction mixture consisted of 2.5%
xylan, 50 mM sodium acetate (AcONa) buffer (pH 5) and the appropriate dilution of the purified enzyme or culture crude extract. Standard assays were incubated at 50 ºC and 1,200 rpm for 5 and 10 min, in order to check the linearity of the activity.
Enzymatic activity against glucose-containing substrates was measured following the release of glucose using the Glucose-TR kit (Spinreact, Vall d'en Bas, Spain) according to the manufacturer's instructions.
The BCA method was used to quantify proteins, using bovine serum albumin (BSA) as standard and Pierce reagents (Thermo scientific, Waltham, MA, USA), following the manufacturer's instructions.
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