dispase  (Worthington Biochemical)

Journal: Journal of Virology

Article Title: Conformational Changes in the Adenovirus Hexon Subunit Responsible for Regulating Cytoplasmic Dynein Recruitment

doi: 10.1128/JVI.02889-14

Figure Lengend Snippet: Dispase treatment reduces capsid redistribution to the nucleus. (A) pH-dependent dispase cleavage of AdV5 capsid hexon. Purified AdV5 capsids were treated with dispase at pH 7.4 or pH 4.4 or kept at pH 4.4 alone before being boiled in PSB and subjected

Article Snippet: For controlled proteolysis, hexon or AdV capsids were incubated with a 6-fold molar excess of dispase (neutral protease; Worthington Biochemicals, Freehold, NJ) over hexon in dispase buffer (100 mM Trizma-maleate, 120 mM NaCl, 2.5 mM CaCl2 ), and the pH was adjusted to either pH 7.4 (neutral) or pH 4.4 (acidic) for 5 min at 37°C until the reaction was stopped by the addition of either protein sample buffer (PSB) or 5 mM EDTA to the mixture.

Techniques: Purification

Journal: Journal of Virology

Article Title: Conformational Changes in the Adenovirus Hexon Subunit Responsible for Regulating Cytoplasmic Dynein Recruitment

doi: 10.1128/JVI.02889-14

Figure Lengend Snippet: Dispase hydrolysis inhibits low-pH effects on hexon structure and dynein binding. (A) Purified hexon was tested for dissociation after dispase treatment. Dispase exposure of hexon at low pH resulted in two major digestion products (85 kDa [I] and 15 kDa

Article Snippet: For controlled proteolysis, hexon or AdV capsids were incubated with a 6-fold molar excess of dispase (neutral protease; Worthington Biochemicals, Freehold, NJ) over hexon in dispase buffer (100 mM Trizma-maleate, 120 mM NaCl, 2.5 mM CaCl2 ), and the pH was adjusted to either pH 7.4 (neutral) or pH 4.4 (acidic) for 5 min at 37°C until the reaction was stopped by the addition of either protein sample buffer (PSB) or 5 mM EDTA to the mixture.

Techniques: Binding Assay, Purification

Journal: Journal of Virology

Article Title: Conformational Changes in the Adenovirus Hexon Subunit Responsible for Regulating Cytoplasmic Dynein Recruitment

doi: 10.1128/JVI.02889-14

Figure Lengend Snippet: The AdV5 hexon HVR1 mutant is inhibited in dynein binding and cytoplasmic transport. (A) Dispase insensitivity of mutant hexon. Immunopurified hexons of wild-type (WT) AdV5, AdV5HVR48(1), and WT AdV48 were tested for dissociation after dispase treatment.

Article Snippet: For controlled proteolysis, hexon or AdV capsids were incubated with a 6-fold molar excess of dispase (neutral protease; Worthington Biochemicals, Freehold, NJ) over hexon in dispase buffer (100 mM Trizma-maleate, 120 mM NaCl, 2.5 mM CaCl2 ), and the pH was adjusted to either pH 7.4 (neutral) or pH 4.4 (acidic) for 5 min at 37°C until the reaction was stopped by the addition of either protein sample buffer (PSB) or 5 mM EDTA to the mixture.

Techniques: Mutagenesis, Binding Assay

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: SMAD3 Deficiency Promotes Inflammatory Aortic Aneurysms in Angiotensin II-Infused Mice Via Activation of iNOS

doi: 10.1161/JAHA.113.000269

Figure Lengend Snippet: Defective contractile function of S3KO aorta. A through C, PE‐induced contraction of WT and S3KO aortic rings treated with placebo (A), 200 μmol/L l‐NAME (B), or 200 μmol/L aminoguanidine (C) (n=4 per group; duplicate experiments were performed). Contraction was measured at PE concentrations ranging from 10 −10 to 10 −3 mol/L, and concentration–response curves were constructed. Note that contraction is expressed as the percentage of maximal contraction induced by incubation of aortic rings in 80 mmol/L high potassium (Hi‐K + ) buffer. For placebo and l‐NAME treatment, endothelium‐intact aortic segments were used, whereas endothelium‐denuded aortas were used in aminoguanidine treatment to eliminate the vasorelaxant effect of eNOS‐derived NO. D, pEC 50 of WT and S3KO aortas in response to PE, indicating tissue sensitivity toward the drug. Note that pEC 50 is the negative logarithm of EC 50 , which refers to the concentration of a drug that induces a half‐maximal response. E, Maximal contraction force generated when incubated with 80 mmol/L Hi‐K + buffer. F, Aortic mRNA level of AngII type 1a (AT1a), type 1b (AT1b), and type 2 (AT2) receptors in WT and S3KO mice (n=3 per group; triplicate experiments were performed). Gene expression was normalized to RPL27. G through I, AngII‐induced contraction in ex vivo aortic segments treated with placebo (G), 200 μmol/L l‐NAME (H), or 200 μmol/L aminoguanidine (I) (n=5 per group; duplicate experiments were performed). Note that AngII elicited a rapid and transient contraction of infrarenal aortic segments at a concentration of 1 μg/mL, whereas minimal (

Article Snippet: Briefly, the whole aorta was dissected out from its origin at the left ventricle to the iliac bifurcation, denuded of periaortic fat, cut into small pieces (≈2‐mm segments), and incubated in 5 mL of Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (#16000036; Life Technologies) and 1.36 mg/mL of type II collagenase (#LS004174; Worthington Biochemical Corporation) in agitation at 115 rpm for 2 hours at 37°C.

Techniques: Concentration Assay, Construct, Incubation, Derivative Assay, Generated, Mouse Assay, Expressing, Ex Vivo

Journal: Nature methods

Article Title: The proteomes of transcription factories containing RNA polymerases I, II or III

doi: 10.1038/nmeth.1705

Figure Lengend Snippet: Purification procedure. (a ) Strategy. Cartoon (top left): chromatin loop with nucleosomes (green circles) tethered to a polymerizing complex (oval) attached to the substructure (brown). Cells are permeabilized, in some cases a run-on performed in [ 32 P]UTP so nascent RNA can be tracked, nuclei are washed with NP40, most chromatin detached with a nuclease (here, DNase I), chromatin-depleted nuclei resuspended in NLB, and polymerizing complexes released from the substructure with caspases. After pelleting, chromatin associated with polymerizing complexes in the supernatant is degraded with DNase I, and complexes partially resolved in 2D gels (using “blue native” and “native” gels in the first and second dimensions); rough positions of complexes (and a control region, c) are shown. Finally, different regions are excised, and their content analyzed by mass spectrometry. ( b ) Recovery of [ 32 P]RNA, after including a “run-on”. Fractions correspond to those at the same level in ( a ). ( c) “Run-on” activity assayed later during fractionation (as in a , but without run-on at beginning). Different fractions, with names as in ( a ), were allowed to extend transcripts by

Article Snippet: Resuspended nuclei were digested (30 min; 33°C) with either (i) DNase I (protease- and RNase-free; Worthington;10 units/107 cells in 100 μl PB plus 0.5 mM CaCl2 ), or (ii) Hae III (Invitrogen; 1000 units/107 cells), or (iii) Hind III (New England Biolabs; 1000 units/107 cells) in PB; reactions were stopped by adding EDTA to 2.5 mM and cooling in iced water.

Techniques: Purification, Mass Spectrometry, Activity Assay, Fractionation

Journal: Nucleic Acids Research

Article Title: An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

doi: 10.1093/nar/gkp732

Figure Lengend Snippet: LC-MS analysis of RNaseT1 digests of yeast tRNA Phe−1 . ( a ) Base peak chromatogram of the RNase T1 digest of the yeast tRNA Phe−1 preparation (200 fmol). Chromatography was performed as described in the ‘Materials and Methods’ section. Sixteen major oligoribonucleotide peaks, indicated by arrows with peak numbers, are assigned to the fragments of yeast tRNA Phe−1 , tRNA Phe−2 , tRNA Lys−2 , or tRNA Tyr (see Table 1 ). ( b and c ) A typical MS spectrum of the RNase T1 digest of tRNA Phe−1 ; (b) [AUUUAm 2 G > p] 2− and (c) [ACmUGmAAyWAΨUm 5 CUG > p] 3− .

Article Snippet: RNase T1 was purchased from Worthington (Lakewood, NJ) and further purified by reversed-phase liquid chromatography (RPLC) before use.

Techniques: Liquid Chromatography with Mass Spectroscopy, Chromatography, Mass Spectrometry

Journal: Nucleic Acids Research

Article Title: An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

doi: 10.1093/nar/gkp732

Figure Lengend Snippet: CID-MS/MS spectrum of [AUUUAm 2 G] 2− derived from RNase T1 digestion of yeast tRNA Phe−1 . The doubly charged ion with m / z = 966.6 was analyzed by CID. The nucleotide sequence was verified by manual interpretation of the a- and c-type (normal text) and w - and y -type ( italic text ) product ion series as indicated in the figure. The parent ion losing methyl guanine [P−B(mG) 2− ], the parent ion losing adenine [P−B(A) 2− ], the y5 ion losing methyl guanine [y5−B(mG) 2− ], the y5 ion losing adenine [y5−B(A) 2− ], y5 2− and c5 2− were doubly charged products. All other assigned signals were singly charged products, unless indicated otherwise. The asterisks indicate hydrated or dehydrated ions of a-, c-, w- or y-type products.

Article Snippet: RNase T1 was purchased from Worthington (Lakewood, NJ) and further purified by reversed-phase liquid chromatography (RPLC) before use.

Techniques: Mass Spectrometry, Derivative Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

doi: 10.1093/nar/gkp732

Figure Lengend Snippet: Base peak chromatogram of the RNaseT1 digest of yeast U4 snRNA isolated from the Lsm3-associated RNP complex. The gel piece containing U4 snRNA was in-gel digested with RNaseT1 and subjected to the LC-MS analysis. Major oligoribonucleotide peaks assigned as RNase T1 fragments of yeast U4 snRNA are indicated by arrows with the corresponding sequence. Detailed data for MS/MS-based assignment of each fragment are given in Supplementary Table S3 .

Article Snippet: RNase T1 was purchased from Worthington (Lakewood, NJ) and further purified by reversed-phase liquid chromatography (RPLC) before use.

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Sequencing, Mass Spectrometry