Structured Review

Millipore protk
Fig. 7. Analysis of Dicer cleavage reactions on native ( A ) and denaturing ( B ) gels, and demonstration that complexes X, Y and Z contain siRNA products ( C ). (A and B) Increasing amounts of Dicer-HisC (0, 10, 30 and 100 ng), either native (lanes 1–8) or pre-incubated with <t>ProtK</t> beads (lanes 9–12), were incubated with 1 fmol of the 32 P-labelled 130 or 70 bp dsRNA at <t>37°C</t> for 30 min. Halves of each reaction were analysed on two different gel types. Lanes S and D, ss and ds 21 nt siRNA markers. The percentage of radioactivity representing siRNA products is indicated below each lane. (C) Lanes from the non-denaturating gel (shown at the top), representing analysis of reactions containing 70 bp dsRNA and 100 ng of either proteolysed (lanes 1–9) or native (lanes 10–18) Dicer, were cut into 1 cm pieces. RNA was eluted, ethanol precipitated with a tRNA carrier and electrophoresed on an 8 M urea gel. Positions of complexes X, Y and Z are indicated.
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Images

1) Product Images from "Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP"

Article Title: Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP

Journal: The EMBO Journal

doi: 10.1093/emboj/cdf582

Fig. 7. Analysis of Dicer cleavage reactions on native ( A ) and denaturing ( B ) gels, and demonstration that complexes X, Y and Z contain siRNA products ( C ). (A and B) Increasing amounts of Dicer-HisC (0, 10, 30 and 100 ng), either native (lanes 1–8) or pre-incubated with ProtK beads (lanes 9–12), were incubated with 1 fmol of the 32 P-labelled 130 or 70 bp dsRNA at 37°C for 30 min. Halves of each reaction were analysed on two different gel types. Lanes S and D, ss and ds 21 nt siRNA markers. The percentage of radioactivity representing siRNA products is indicated below each lane. (C) Lanes from the non-denaturating gel (shown at the top), representing analysis of reactions containing 70 bp dsRNA and 100 ng of either proteolysed (lanes 1–9) or native (lanes 10–18) Dicer, were cut into 1 cm pieces. RNA was eluted, ethanol precipitated with a tRNA carrier and electrophoresed on an 8 M urea gel. Positions of complexes X, Y and Z are indicated.
Figure Legend Snippet: Fig. 7. Analysis of Dicer cleavage reactions on native ( A ) and denaturing ( B ) gels, and demonstration that complexes X, Y and Z contain siRNA products ( C ). (A and B) Increasing amounts of Dicer-HisC (0, 10, 30 and 100 ng), either native (lanes 1–8) or pre-incubated with ProtK beads (lanes 9–12), were incubated with 1 fmol of the 32 P-labelled 130 or 70 bp dsRNA at 37°C for 30 min. Halves of each reaction were analysed on two different gel types. Lanes S and D, ss and ds 21 nt siRNA markers. The percentage of radioactivity representing siRNA products is indicated below each lane. (C) Lanes from the non-denaturating gel (shown at the top), representing analysis of reactions containing 70 bp dsRNA and 100 ng of either proteolysed (lanes 1–9) or native (lanes 10–18) Dicer, were cut into 1 cm pieces. RNA was eluted, ethanol precipitated with a tRNA carrier and electrophoresed on an 8 M urea gel. Positions of complexes X, Y and Z are indicated.

Techniques Used: Incubation, Radioactivity

2) Product Images from "Enrichment of Sialylated IgG by Lectin Fractionation Does Not Enhance the Efficacy of Immunoglobulin G in a Murine Model of Immune Thrombocytopenia"

Article Title: Enrichment of Sialylated IgG by Lectin Fractionation Does Not Enhance the Efficacy of Immunoglobulin G in a Murine Model of Immune Thrombocytopenia

Journal: PLoS ONE

doi: 10.1371/journal.pone.0021246

Measurement of the degree of sialylation by SNA lectin inhibition ELISA. % SNA lectin binding after pre-incubation A ) with IVIg, IVIg-SA (+) or IVIg-SA (−) B ) with Fab and Fc fragments derived from IVIg, IVIg-SA (+) or IVIg-SA (−) C ) with Fab or protK digested Fab fragments derived from IVIg, IVIg-SA (+) or IVIg-SA (−) D ) with Fc or protK digested Fc fragments derived from IVIg, IVIg-SA (+) or IVIg-SA (−). Data represents mean ± SEM, n = 3.
Figure Legend Snippet: Measurement of the degree of sialylation by SNA lectin inhibition ELISA. % SNA lectin binding after pre-incubation A ) with IVIg, IVIg-SA (+) or IVIg-SA (−) B ) with Fab and Fc fragments derived from IVIg, IVIg-SA (+) or IVIg-SA (−) C ) with Fab or protK digested Fab fragments derived from IVIg, IVIg-SA (+) or IVIg-SA (−) D ) with Fc or protK digested Fc fragments derived from IVIg, IVIg-SA (+) or IVIg-SA (−). Data represents mean ± SEM, n = 3.

Techniques Used: Inhibition, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Derivative Assay

Related Articles

Radioactivity:

Article Title: Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP
Article Snippet: BSA (10 µg) was additionally added to reactions in the experiment shown in Figure C. For proteolytic activation, reactions were pre-incubated for 10 min at 37°C, under assay conditions, with the indicated amounts of ProtK (Sigma) prior to dsRNA addition, or 1 µg of Dicer-HisC was pre-incubated with ProtK–agarose beads (Sigma; 3 mg containing 0.8 U of ProtK, for 5 min at 37°C). .. For quantification, a percentage of radioactivity corresponding to siRNA products was calculated in relation to either the input or a sum of the bands, representing the substrate, intermediates and siRNA products present in individual lanes.

Activation Assay:

Article Title: Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP
Article Snippet: .. BSA (10 µg) was additionally added to reactions in the experiment shown in Figure C. For proteolytic activation, reactions were pre-incubated for 10 min at 37°C, under assay conditions, with the indicated amounts of ProtK (Sigma) prior to dsRNA addition, or 1 µg of Dicer-HisC was pre-incubated with ProtK–agarose beads (Sigma; 3 mg containing 0.8 U of ProtK, for 5 min at 37°C). .. Reactions were analysed on 10% polyacrylamide/8 M urea gels, which were processed for autoradiography or quantification, using the Storm 860 PhosphorImager (Molecular Dynamics).

ATP Assay:

Article Title: Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP
Article Snippet: We have verified, using P19 cell extracts and a bioluminescence ATP assay kit (Sigma), that the procedure with 10 mM glucose decreases ATP to < 100 nM. .. BSA (10 µg) was additionally added to reactions in the experiment shown in Figure C. For proteolytic activation, reactions were pre-incubated for 10 min at 37°C, under assay conditions, with the indicated amounts of ProtK (Sigma) prior to dsRNA addition, or 1 µg of Dicer-HisC was pre-incubated with ProtK–agarose beads (Sigma; 3 mg containing 0.8 U of ProtK, for 5 min at 37°C).

Autoradiography:

Article Title: Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP
Article Snippet: BSA (10 µg) was additionally added to reactions in the experiment shown in Figure C. For proteolytic activation, reactions were pre-incubated for 10 min at 37°C, under assay conditions, with the indicated amounts of ProtK (Sigma) prior to dsRNA addition, or 1 µg of Dicer-HisC was pre-incubated with ProtK–agarose beads (Sigma; 3 mg containing 0.8 U of ProtK, for 5 min at 37°C). .. Reactions were analysed on 10% polyacrylamide/8 M urea gels, which were processed for autoradiography or quantification, using the Storm 860 PhosphorImager (Molecular Dynamics).

Incubation:

Article Title: Abnormal differentiation of dopaminergic neurons in zebrafish trpm7 mutant larvae impairs development of the motor pattern
Article Snippet: .. Whole-mount larvae were washed out of fixative with double-distilled water (ddH2 O), permeabilized by either 5 hrs incubation at room temperature in ddH2 O or 30 min treatment with 40 mg/mL ProtK, blocked in PBDT (Phosphate buffered solution, 2.5 % Goat serum, 1 % bovine serum albumin, 1 % DMSO, 0.5 % Tritonx-100) for 1 hr, and incubated in primary antibody [mouse anti-tyrosine hydroxylase (1:500, Sigma-Aldrich) or rabbit anti- 5HT] diluted in PBDT overnight at 4°C. .. In experiments where we used the peroxidase anti-peroxidase system (Jackson Immunoresearch Laboratories, West Grove, PA), we subsequently incubated embryos in 2° goat anti-mouse antibodies and 3° mPAP antibody.

Article Title: Enrichment of Sialylated IgG by Lectin Fractionation Does Not Enhance the Efficacy of Immunoglobulin G in a Murine Model of Immune Thrombocytopenia
Article Snippet: Proteinase K digestion of Fc fragments To determine the degree of Fc sialylation, Fc fragments derived from IVIg, IVIg-SA (+) or IVIg-SA (−) were digested with protK (proteinase K, Sigma-Aldrich, St. Louis, MO, USA). .. The Fc fragments were overnight dialyzed against PBS at 4°C, followed by incubation with protK (ratio 0.6 U/mg protein, overnight, at 50°C).

Microscopy:

Article Title: Abnormal differentiation of dopaminergic neurons in zebrafish trpm7 mutant larvae impairs development of the motor pattern
Article Snippet: Whole-mount larvae were washed out of fixative with double-distilled water (ddH2 O), permeabilized by either 5 hrs incubation at room temperature in ddH2 O or 30 min treatment with 40 mg/mL ProtK, blocked in PBDT (Phosphate buffered solution, 2.5 % Goat serum, 1 % bovine serum albumin, 1 % DMSO, 0.5 % Tritonx-100) for 1 hr, and incubated in primary antibody [mouse anti-tyrosine hydroxylase (1:500, Sigma-Aldrich) or rabbit anti- 5HT] diluted in PBDT overnight at 4°C. .. Whole-mount larvae were alternatively prepared using Alexa 488 conjugated secondary antibodies and optically sectioned using a Zeiss 700 confocal microscope and ZEN 2010 software (Carl Zeiss Inc., Germany).

Purification:

Article Title: Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP
Article Snippet: For ATP depletion, reactions were pre-incubated for 20 min at 37°C with 2 mM (for purified Dicer) or 10 mM (for IPs) glucose and 1 U of hexokinase (HK, Sigma), or with 1 U of CIP (Roche). .. BSA (10 µg) was additionally added to reactions in the experiment shown in Figure C. For proteolytic activation, reactions were pre-incubated for 10 min at 37°C, under assay conditions, with the indicated amounts of ProtK (Sigma) prior to dsRNA addition, or 1 µg of Dicer-HisC was pre-incubated with ProtK–agarose beads (Sigma; 3 mg containing 0.8 U of ProtK, for 5 min at 37°C).

Concentration Assay:

Article Title: Enrichment of Sialylated IgG by Lectin Fractionation Does Not Enhance the Efficacy of Immunoglobulin G in a Murine Model of Immune Thrombocytopenia
Article Snippet: Proteinase K digestion of Fc fragments To determine the degree of Fc sialylation, Fc fragments derived from IVIg, IVIg-SA (+) or IVIg-SA (−) were digested with protK (proteinase K, Sigma-Aldrich, St. Louis, MO, USA). .. After the addition of sodium azide (NaN3 ) with a final concentration of 0.05% the digested Fc fragments were stored at 4°C.

Generated:

Article Title: Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP
Article Snippet: BSA (10 µg) was additionally added to reactions in the experiment shown in Figure C. For proteolytic activation, reactions were pre-incubated for 10 min at 37°C, under assay conditions, with the indicated amounts of ProtK (Sigma) prior to dsRNA addition, or 1 µg of Dicer-HisC was pre-incubated with ProtK–agarose beads (Sigma; 3 mg containing 0.8 U of ProtK, for 5 min at 37°C). .. For calculation of molar ratios of Dicer to generated siRNA products, the Dicer-HisC purity was assumed to be 75%.

Activity Assay:

Article Title: Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP
Article Snippet: Paragraph title: Assays of Dicer activity ... BSA (10 µg) was additionally added to reactions in the experiment shown in Figure C. For proteolytic activation, reactions were pre-incubated for 10 min at 37°C, under assay conditions, with the indicated amounts of ProtK (Sigma) prior to dsRNA addition, or 1 µg of Dicer-HisC was pre-incubated with ProtK–agarose beads (Sigma; 3 mg containing 0.8 U of ProtK, for 5 min at 37°C).

Activated Clotting Time Assay:

Article Title: Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP
Article Snippet: If not indicated otherwise, reactions contained 3–5 fmol of dsRNA internally labelled with [α-32 P]UTP (final sp. act. .. BSA (10 µg) was additionally added to reactions in the experiment shown in Figure C. For proteolytic activation, reactions were pre-incubated for 10 min at 37°C, under assay conditions, with the indicated amounts of ProtK (Sigma) prior to dsRNA addition, or 1 µg of Dicer-HisC was pre-incubated with ProtK–agarose beads (Sigma; 3 mg containing 0.8 U of ProtK, for 5 min at 37°C).

Derivative Assay:

Article Title: Enrichment of Sialylated IgG by Lectin Fractionation Does Not Enhance the Efficacy of Immunoglobulin G in a Murine Model of Immune Thrombocytopenia
Article Snippet: .. Proteinase K digestion of Fc fragments To determine the degree of Fc sialylation, Fc fragments derived from IVIg, IVIg-SA (+) or IVIg-SA (−) were digested with protK (proteinase K, Sigma-Aldrich, St. Louis, MO, USA). .. The Fc fragments were overnight dialyzed against PBS at 4°C, followed by incubation with protK (ratio 0.6 U/mg protein, overnight, at 50°C).

Software:

Article Title: Abnormal differentiation of dopaminergic neurons in zebrafish trpm7 mutant larvae impairs development of the motor pattern
Article Snippet: Whole-mount larvae were washed out of fixative with double-distilled water (ddH2 O), permeabilized by either 5 hrs incubation at room temperature in ddH2 O or 30 min treatment with 40 mg/mL ProtK, blocked in PBDT (Phosphate buffered solution, 2.5 % Goat serum, 1 % bovine serum albumin, 1 % DMSO, 0.5 % Tritonx-100) for 1 hr, and incubated in primary antibody [mouse anti-tyrosine hydroxylase (1:500, Sigma-Aldrich) or rabbit anti- 5HT] diluted in PBDT overnight at 4°C. .. Whole-mount larvae were alternatively prepared using Alexa 488 conjugated secondary antibodies and optically sectioned using a Zeiss 700 confocal microscope and ZEN 2010 software (Carl Zeiss Inc., Germany).