rat anti zp2  (Santa Cruz Biotechnology)

Journal: PLoS ONE

Article Title: Amyloid Properties of the Mouse Egg Zona Pellucida

doi: 10.1371/journal.pone.0129907

Figure Lengend Snippet: A) Thirty isolated ZP were treated with (+) or without (-) DMSO followed by incubation with the PAD ligand and eluted proteins examined by immunoblot using anti-ZP1, ZP2, and ZP3 antibodies. ZP, an equivalent number of ZP placed directly in SDS-PAGE loading buffer without PAD incubation. Buffer, PBS only incubated with PAD beads. Molecular weight markers indicate kDa. B) phase images of oocytes incubated in the presence (+) or absence (no DMSO) of 90% DMSO for 2 minutes at room temperature. Arrow indicates ZP. Scale bar = 50 μm. C) Equal numbers of ZP were exposed to 90% DMSO, 1% SDS or 0.25% SDS prior to spotting on nitrocellulose using a dot blot apparatus. Membranes were then incubated with the amyloid anti-fibrillar OC and anti-oligomer A11 antibodies. Dot blots were rehybridized with anti-ZP2 antibody to confirm the presence of ZP protein in each well. b, buffer.

Article Snippet: Samples were electro blotted onto a polyvinylidene difluoride membrane (cat. no. IPVH00010, EMD Millipore, Bedford, MA) and the membrane was blocked for 1 h at RT with shaking in 3% nonfat dry milk in TBST and then incubated overnight with shaking at 4°C with either 0.5 μg/ml rat anti-mouse ZP1 (M1.4, sc-32751, Santa Cruz Biotechnology, Santa Cruz, CA), 0.25 μg/ml rat anti-mouse ZP2 (IE-3, sc-32752, Santa Cruz Biotechnology), or 0.5 μg/ml rabbit anti-human ZP3 (H-300, sc-25802, Santa Cruz Biotechnology) in 3% nonfat dry milk in TBST.

Techniques: Isolation, Incubation, Western Blot, SDS Page, Molecular Weight, Dot Blot

Journal: PLoS ONE

Article Title: Amyloid Properties of the Mouse Egg Zona Pellucida

doi: 10.1371/journal.pone.0129907

Figure Lengend Snippet: A) Schematic diagram of mature ZP1, ZP2, and ZP3 with amyloidogenic regions predicted by AmylPred 2 indicated as red bars above the individual domains. Yellow box, trefoil domain. Numbers signify amino acid number. B) Structure based sequence alignment of the ZP polymerization domain of mouse ZP1 (aa 268–541), ZP2 (aa 361–630), and ZP3 (aa 42–305) showing amyloidogenic sites (blue highlighting), as predicted by the Amylpred2 algorithm. Cysteine residues are noted by black boxes. The internal hydrophobic patch (IHP) is indicated by a red box . The β-strand secondary structure based on the crystal structure of chicken ZP3 is noted by orange bars (ZP-N subdomain) and green bars (ZP-C subdomain) above the amino acid sequences . C) Structure based alignment of ZP-N domains in mouse ZP1 (N1, N2), ZP2 (N1-N4), ZP3 (N), abalone VERL repeat 10 (R10) and yeast α-agglutinin/Sag 1p showing predicted amyloidogenic sites in blue highlighting as determined by the AmylPred2 algorithm. The β-strand secondary structure, based on structure of mZP-N is indicated by orange bars above the amino acid sequence . Cysteine residues are noted by black boxes. * , indicate sites essential or important for ZP2-sperm and α-agglutinin-a-agglutinin binding [ , ].

Article Snippet: Samples were electro blotted onto a polyvinylidene difluoride membrane (cat. no. IPVH00010, EMD Millipore, Bedford, MA) and the membrane was blocked for 1 h at RT with shaking in 3% nonfat dry milk in TBST and then incubated overnight with shaking at 4°C with either 0.5 μg/ml rat anti-mouse ZP1 (M1.4, sc-32751, Santa Cruz Biotechnology, Santa Cruz, CA), 0.25 μg/ml rat anti-mouse ZP2 (IE-3, sc-32752, Santa Cruz Biotechnology), or 0.5 μg/ml rabbit anti-human ZP3 (H-300, sc-25802, Santa Cruz Biotechnology) in 3% nonfat dry milk in TBST.

Techniques: Sequencing, Binding Assay

Journal: The Journal of reproduction and development

Article Title: Functional characterization of double-knockout mouse sperm lacking SPAM1 and ACR or SPAM1 and PRSS21 in fertilization.

doi: 10.1262/jrd.2011-006

Figure Lengend Snippet: Fig. 3. Penetration assays of epididymal sperm through the cumulus. Epididymal sperm from wild-type (WT), Spam1-/- (SPAM1 KO), Spam1-/-/Acr-/- (S/A DKO) and Spam1-/-/Prss21-/- (S/P DKO) mice were labeled with Hoechst 33342, capacitated and mixed with OCCs at a concentration of 150 sperm/μl. After incubation for 15 or 30 min, the cumulus and sperm were fixed with paraformaldehyde. The oocyte ZPs were visualized by immunostaining with anti-ZP2 monoclonal antibody (green colors). Hoechst-labeled sperm (red dots) within the cumulus matrix (CM) and on the oocyte ZP were observed under a microscope (A) and counted (B). Bright-field and fluorescence images were acquired, processed by the MetaMorph software and then merged into one picture. Only pictures obtained 30 min after insemination are shown. A total of 20–24 oocytes were examined for WT, SPAM1 KO, S/A DKO, and S/P DKO sperm (n=4). The differences in the numbers of sperm in the cumulus matrix and on the ZP surface at 15 and 30 min after insemination are significant between WT and SPAM1 KO, S/A DKO or S/P DKO sperm and between SPAM1 KO and S/A or S/P DKO sperm (P<0.001), except between SPAM1 KO and S/A DKO sperm in the cumulus matrix at the 15-min stage (P=0.7). Bar=100 μm.

Article Snippet: Rat monoclonal antibody against mouse ZP2 (IE-3, SC-32752) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Labeling, Concentration Assay, Incubation, Immunostaining, Microscopy, Fluorescence, Software