Structured Review

Millipore in vitro cleavage assay zonae pellucidae
Cleavage of ZP2 by ovastacin. (A) A schematic of mouse ZP2 with eight diacidic motifs (triangles), the initial cleavage site of ovastacin, the binding sites of mAbs IE-3 and M2c.2 N terminal and C terminal to the initial cleavage site, and the localization of the single N -glycan in the N terminus. The potential size of peptides resulting from cleavage of the N-terminal fragment with ovastacin detected by IE-3 is shown on the bottom. (B) Immunoblot of eggs (15) or two-cell embryos (15) from normal and Astl Null females stained with IE-3 mAb that detects N-terminal fragments of ZP2. The top bands (120 kD) are intact ZP2, and bottom bands are a heterogeneous mixture of cleaved N-terminal fragments. Arrows indicate the four peptides detected on the immunoblot probed with IE-3. (C) Isolated <t>zonae</t> <t>pellucidae</t> (15–20) were incubated with partially purified recombinant ovastacin (Ovastacin Rec ) at 37°C for 0, 2, 4, and 8 h. (D) Same as in C but with insect cell supernatant lacking (control [Ctrl]) or containing (Ovst Rec ) recombinant ovastacin after 4 h of incubation with zonae pellucidae from normal (Norm) or Astl Null eggs. (C and D) Cleavage was detected by immunoblotting with M2c.2 to detect the ZP2 C-terminal fragment.
In Vitro Cleavage Assay Zonae Pellucidae, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy"

Article Title: Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201112094

Cleavage of ZP2 by ovastacin. (A) A schematic of mouse ZP2 with eight diacidic motifs (triangles), the initial cleavage site of ovastacin, the binding sites of mAbs IE-3 and M2c.2 N terminal and C terminal to the initial cleavage site, and the localization of the single N -glycan in the N terminus. The potential size of peptides resulting from cleavage of the N-terminal fragment with ovastacin detected by IE-3 is shown on the bottom. (B) Immunoblot of eggs (15) or two-cell embryos (15) from normal and Astl Null females stained with IE-3 mAb that detects N-terminal fragments of ZP2. The top bands (120 kD) are intact ZP2, and bottom bands are a heterogeneous mixture of cleaved N-terminal fragments. Arrows indicate the four peptides detected on the immunoblot probed with IE-3. (C) Isolated zonae pellucidae (15–20) were incubated with partially purified recombinant ovastacin (Ovastacin Rec ) at 37°C for 0, 2, 4, and 8 h. (D) Same as in C but with insect cell supernatant lacking (control [Ctrl]) or containing (Ovst Rec ) recombinant ovastacin after 4 h of incubation with zonae pellucidae from normal (Norm) or Astl Null eggs. (C and D) Cleavage was detected by immunoblotting with M2c.2 to detect the ZP2 C-terminal fragment.
Figure Legend Snippet: Cleavage of ZP2 by ovastacin. (A) A schematic of mouse ZP2 with eight diacidic motifs (triangles), the initial cleavage site of ovastacin, the binding sites of mAbs IE-3 and M2c.2 N terminal and C terminal to the initial cleavage site, and the localization of the single N -glycan in the N terminus. The potential size of peptides resulting from cleavage of the N-terminal fragment with ovastacin detected by IE-3 is shown on the bottom. (B) Immunoblot of eggs (15) or two-cell embryos (15) from normal and Astl Null females stained with IE-3 mAb that detects N-terminal fragments of ZP2. The top bands (120 kD) are intact ZP2, and bottom bands are a heterogeneous mixture of cleaved N-terminal fragments. Arrows indicate the four peptides detected on the immunoblot probed with IE-3. (C) Isolated zonae pellucidae (15–20) were incubated with partially purified recombinant ovastacin (Ovastacin Rec ) at 37°C for 0, 2, 4, and 8 h. (D) Same as in C but with insect cell supernatant lacking (control [Ctrl]) or containing (Ovst Rec ) recombinant ovastacin after 4 h of incubation with zonae pellucidae from normal (Norm) or Astl Null eggs. (C and D) Cleavage was detected by immunoblotting with M2c.2 to detect the ZP2 C-terminal fragment.

Techniques Used: Binding Assay, Staining, Isolation, Incubation, Purification, Recombinant

Related Articles

In Vitro:

Article Title: Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy
Article Snippet: The fractions with the highest concentration of ovastacin were used in subsequent assays. .. In vitro cleavage assay Zonae pellucidae were isolated from 150 oocytes by freeze thawing four times in 100 µl PBS, pH 7.4, 0.1% IGEPAL CA-630 (Sigma-Aldrich), and 0.5 M NaCl. .. Isolated zonae were solubilized in 30 µl PBS, 0.4% PVP, and 0.1% SDS by heating at 60°C for 30 min. Solubilized zona samples were incubated with recombinant ovastacin at 37°C over time (0–8 h), and cleavage was analyzed by immunoblotting with mAb M2c.2.

Cleavage Assay:

Article Title: Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy
Article Snippet: The fractions with the highest concentration of ovastacin were used in subsequent assays. .. In vitro cleavage assay Zonae pellucidae were isolated from 150 oocytes by freeze thawing four times in 100 µl PBS, pH 7.4, 0.1% IGEPAL CA-630 (Sigma-Aldrich), and 0.5 M NaCl. .. Isolated zonae were solubilized in 30 µl PBS, 0.4% PVP, and 0.1% SDS by heating at 60°C for 30 min. Solubilized zona samples were incubated with recombinant ovastacin at 37°C over time (0–8 h), and cleavage was analyzed by immunoblotting with mAb M2c.2.

Isolation:

Article Title: Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy
Article Snippet: The fractions with the highest concentration of ovastacin were used in subsequent assays. .. In vitro cleavage assay Zonae pellucidae were isolated from 150 oocytes by freeze thawing four times in 100 µl PBS, pH 7.4, 0.1% IGEPAL CA-630 (Sigma-Aldrich), and 0.5 M NaCl. .. Isolated zonae were solubilized in 30 µl PBS, 0.4% PVP, and 0.1% SDS by heating at 60°C for 30 min. Solubilized zona samples were incubated with recombinant ovastacin at 37°C over time (0–8 h), and cleavage was analyzed by immunoblotting with mAb M2c.2.

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    Millipore in vitro cleavage assay zonae pellucidae
    Cleavage of ZP2 by ovastacin. (A) A schematic of mouse ZP2 with eight diacidic motifs (triangles), the initial cleavage site of ovastacin, the binding sites of mAbs IE-3 and M2c.2 N terminal and C terminal to the initial cleavage site, and the localization of the single N -glycan in the N terminus. The potential size of peptides resulting from cleavage of the N-terminal fragment with ovastacin detected by IE-3 is shown on the bottom. (B) Immunoblot of eggs (15) or two-cell embryos (15) from normal and Astl Null females stained with IE-3 mAb that detects N-terminal fragments of ZP2. The top bands (120 kD) are intact ZP2, and bottom bands are a heterogeneous mixture of cleaved N-terminal fragments. Arrows indicate the four peptides detected on the immunoblot probed with IE-3. (C) Isolated <t>zonae</t> <t>pellucidae</t> (15–20) were incubated with partially purified recombinant ovastacin (Ovastacin Rec ) at 37°C for 0, 2, 4, and 8 h. (D) Same as in C but with insect cell supernatant lacking (control [Ctrl]) or containing (Ovst Rec ) recombinant ovastacin after 4 h of incubation with zonae pellucidae from normal (Norm) or Astl Null eggs. (C and D) Cleavage was detected by immunoblotting with M2c.2 to detect the ZP2 C-terminal fragment.
    In Vitro Cleavage Assay Zonae Pellucidae, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/in vitro cleavage assay zonae pellucidae/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    in vitro cleavage assay zonae pellucidae - by Bioz Stars, 2021-05
    97/100 stars
      Buy from Supplier

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    Cleavage of ZP2 by ovastacin. (A) A schematic of mouse ZP2 with eight diacidic motifs (triangles), the initial cleavage site of ovastacin, the binding sites of mAbs IE-3 and M2c.2 N terminal and C terminal to the initial cleavage site, and the localization of the single N -glycan in the N terminus. The potential size of peptides resulting from cleavage of the N-terminal fragment with ovastacin detected by IE-3 is shown on the bottom. (B) Immunoblot of eggs (15) or two-cell embryos (15) from normal and Astl Null females stained with IE-3 mAb that detects N-terminal fragments of ZP2. The top bands (120 kD) are intact ZP2, and bottom bands are a heterogeneous mixture of cleaved N-terminal fragments. Arrows indicate the four peptides detected on the immunoblot probed with IE-3. (C) Isolated zonae pellucidae (15–20) were incubated with partially purified recombinant ovastacin (Ovastacin Rec ) at 37°C for 0, 2, 4, and 8 h. (D) Same as in C but with insect cell supernatant lacking (control [Ctrl]) or containing (Ovst Rec ) recombinant ovastacin after 4 h of incubation with zonae pellucidae from normal (Norm) or Astl Null eggs. (C and D) Cleavage was detected by immunoblotting with M2c.2 to detect the ZP2 C-terminal fragment.

    Journal: The Journal of Cell Biology

    Article Title: Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy

    doi: 10.1083/jcb.201112094

    Figure Lengend Snippet: Cleavage of ZP2 by ovastacin. (A) A schematic of mouse ZP2 with eight diacidic motifs (triangles), the initial cleavage site of ovastacin, the binding sites of mAbs IE-3 and M2c.2 N terminal and C terminal to the initial cleavage site, and the localization of the single N -glycan in the N terminus. The potential size of peptides resulting from cleavage of the N-terminal fragment with ovastacin detected by IE-3 is shown on the bottom. (B) Immunoblot of eggs (15) or two-cell embryos (15) from normal and Astl Null females stained with IE-3 mAb that detects N-terminal fragments of ZP2. The top bands (120 kD) are intact ZP2, and bottom bands are a heterogeneous mixture of cleaved N-terminal fragments. Arrows indicate the four peptides detected on the immunoblot probed with IE-3. (C) Isolated zonae pellucidae (15–20) were incubated with partially purified recombinant ovastacin (Ovastacin Rec ) at 37°C for 0, 2, 4, and 8 h. (D) Same as in C but with insect cell supernatant lacking (control [Ctrl]) or containing (Ovst Rec ) recombinant ovastacin after 4 h of incubation with zonae pellucidae from normal (Norm) or Astl Null eggs. (C and D) Cleavage was detected by immunoblotting with M2c.2 to detect the ZP2 C-terminal fragment.

    Article Snippet: In vitro cleavage assay Zonae pellucidae were isolated from 150 oocytes by freeze thawing four times in 100 µl PBS, pH 7.4, 0.1% IGEPAL CA-630 (Sigma-Aldrich), and 0.5 M NaCl.

    Techniques: Binding Assay, Staining, Isolation, Incubation, Purification, Recombinant