proteinase k  (Millipore)

Journal: Structural Dynamics

Article Title: Fixed target matrix for femtosecond time-resolved and in situ serial micro-crystallography

doi: 10.1063/1.4928706

Figure Lengend Snippet: (a) Overview of the orientation distribution of 100 μ m thaumatin crystals (number of crystals n = 69), (b) 40–50 μ m thaumatin crystals (n = 102), and (c) 30 μ m proteinase K crystals (n = 165) mounted in crystallography chips of matching sizes (100, 50, and 30 μ m features). The dots in transparent grey represent the beam directions relative to the crystal coordinate system in Lambert equal-area projection. Dark grey indicates overlap of two or more dots. The centers of the diagrams correspond to the tetragonal c axes, the outermost circle to directions in the a,b-plane. Each “observed” direction is replicated according to the point group (422) but, of all symmetry-related directions, only those pointing to the upper hemisphere are shown. In principle, three parameters are required to define the orientation of a crystal, but for the present purpose, the two angles shown in Figure 3 are sufficient. The third angle, representing rotation of the crystal around the beam direction, is superfluous as this parameter determines the orientation of the diffraction pattern in the detector plane, without changing the pattern itself. Figure prepared with R . 33

Article Snippet: In this initial work, we have used commercially available model systems, thaumatin and proteinase K (Sigma Aldrich).

Techniques:

Journal: Infection and Immunity

Article Title: Effective Protective Immunity to Yersinia pestis Infection Conferred by DNA Vaccine Coding for Derivatives of the F1 Capsular Antigen

doi: 10.1128/IAI.71.1.374-383.2003

Figure Lengend Snippet: Generation of vectors for cellular targeting of F1 expression. (A) The three F1 DNA derivatives used in the study are depicted. pCI-F1 expresses the intact bacterial coding sequence of the F1 anti-gen, pCI-deF1 expresses the putatively mature F1 devoid of the bacterial signal sequence, and pCI-E3/F1 expresses F1 fused to the E3 envelope protein of SFV carrying an eukaryotic signal peptide. (B and C) Radiolabeled, in vitro expression products of pCI-F1, pCI-deF1, and pCI-E3/F1. Polypeptides were generated in a TNT system in the presence or absence of canine microsomes. The β-lactamase cDNA was used as the control for signal cleavage. Purified bacterial F1 was used as the reference for migration of the mature gene product. Prestained, 14 C-labeled polypeptides (Rainbow; Pharmacia) served as MW markers. In panel C, translocation of the E3/F1 hybrid into microsomes was examined by proteinase K treatment in the presence or absence of Triton X-100.

Article Snippet: Where indicated, translation products were treated with proteinase K (0.2 mg/ml; Sigma) for 15 min at 4°C in the presence or absence of 1% Triton X-100.

Techniques: Expressing, Sequencing, In Vitro, Generated, Purification, Migration, Labeling, Translocation Assay

Journal: Journal of Antimicrobial Chemotherapy

Article Title: Cephalosporinases associated with outer membrane vesicles released by Bacteroides spp. protect gut pathogens and commensals against β-lactam antibiotics

doi: 10.1093/jac/dku466

Figure Lengend Snippet: OMVs are produced in vivo by B. thetaiotaomicron and display BtCepA on their surface. (a) Electron microscopic photograph of OMVs collected from a sterile compartment after their diffusion through a 0.22 μm membrane from a compartment containing a B. thetaiotaomicron culture (see the Materials and methods section). Scale bar, ∼100 nm. (b) BtCepA and BtMinpp activities measured after treatment of OMVs with proteinase K. The relative activity is the ratio of the activity measured after proteinase K treatment compared with the activity measured without treatment. Dark grey bars, no proteinase K pre-treatment; light grey bars, proteinase K pre-treatment. * P

Article Snippet: Enzyme activity at the surface of OMVs A suspension of 250 μg of vesicles in 0.1 M phosphate/1 mM EDTA buffer (pH 7.0) was incubated for 5 min (for vesicles from B. thetaiotaomicron ) and 1 h (for all other Bacteroides species) at 37°C in the presence of 100 mg/L proteinase K (Sigma-Aldrich).

Techniques: Produced, In Vivo, Diffusion-based Assay, Activity Assay

Journal: Molecular and Cellular Biology

Article Title: Two Independent Internal Ribosome Entry Sites Are Involved in Translation Initiation of Vascular Endothelial Growth Factor mRNA

doi:

Figure Lengend Snippet: UV cross-linking of COS-7 cell proteins on the VEGF mRNA 5′ UTR. (A) Drawing of the different 32 P-labeled RNA probes, obtained from T7 in vitro transcription and corresponding to the complete or parts of the VEGF 5′ UTR mRNA. Relative positions of the 5′ and 3′ ends of each probe are indicated. (B) UV cross-linking experiments performed with probes A to E. S10 COS-7 cell extracts were incubated with 10 6 cpm of the different probes followed by UV irradiation and treatment with RNases A and ONE (see Materials and Methods). The control (Ct) lane corresponds to proteinase K treatment of sample loaded in the first lane. Size markers are indicated. (C) 32 P-labeled probes corresponding to VEGF probe A (complete 5′ UTR) and to EMCV IRES were cross-linked with proteins extracted from S10 COS-7 cells, and the complex was immunoprecipitated with an anti-PTB antibody. The samples were analyzed before (CL) and after (I) immunoprecipitation. The use of a VEGF or EMCV probe is indicated above the lanes. PTB migration is indicated with an arrow.

Article Snippet: The samples were then treated with a mix of RNase ONE (5 U; Promega) and 2.5 μg of RNase A at 37°C for 30 min and, when indicated, with proteinase K (Sigma) at 37°C for 20 min at a final concentration of 1 mg/ml.

Techniques: Labeling, In Vitro, Incubation, Irradiation, Immunoprecipitation, Migration