Thylakoid membranes were prepared as described previously [66 (link)]. For one-dimensional electrophoresis, thylakoid membranes were separated by SDS-PAGE and western blots were performed as described previously [65 (link)]. The isolated thylakoid membranes were separated by BN-PAGE as described [66 (link), 67 (link)]. For two-dimensional SDS-PAGE separation, the excised BN-PAGE lanes were denatured in SDS sample buffer and 5% β-mercaptoethanol and layered onto 12% SDS polyacrylamide gels with 4 M urea. For immunodetection of thylakoid membrane proteins, the separated proteins were transferred to nitrocellulose membranes and probed with the indicated specific antibodies (Agrisera, Vännäs, Sweden). For detecting signals, alkaline-phosphatase-conjugated goat anti-rabbit IgG (Millipore, Darmstadt, Germany) was used as a secondary antibody, and reactions were revealed using an ECL Kit (GE Healthcare, Marlborough, MA, USA). The signals were detected by ImageQuant LAS 4000 mini (GE Healthcare).
Alkaline phosphatase conjugated goat anti rabbit igg
Manufactured by Merck Group
Sourced in United States
Alkaline phosphatase-conjugated goat anti-rabbit IgG is a laboratory reagent used in immunoassays and other biochemical applications. It is a secondary antibody that binds to rabbit primary antibodies and is conjugated with the enzyme alkaline phosphatase. The enzymatic activity of the alkaline phosphatase can be used to detect and quantify the presence of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Ecl kit, by GE Healthcare (1 mentions)
Sm0661, by Thermo Fisher Scientific (1 mentions)
Mini transfer cell, by Bio-Rad (1 mentions)
Anti ctcf, by Cell Signaling Technology (1 mentions)
Anti rad21, by Abcam (1 mentions)
Alkaline phosphatase conjugated goat anti mouse igg, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Semi dry apparatus, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Gradient sds page, by Thermo Fisher Scientific (1 mentions)
Silver stain plus kit, by Bio-Rad (1 mentions)
Hybond c extra nitrocellulose membrane, by GE Healthcare (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Trans blot cell, by Bio-Rad (1 mentions)
Sigmafast bcip nbt, by Merck Group (1 mentions)
Phytosphingosine, by Merck Group (1 mentions)
Sphinganine, by Merck Group (1 mentions)
1 6 diphenyl 1 3 5 hexatriene dph, by Merck Group (1 mentions)
Optiprep, by Merck Group (1 mentions)
18 protocols using alkaline phosphatase conjugated goat anti rabbit igg
1
Thylakoid Membrane Protein Analysis
2
Purification and Characterization of Recombinant Chitinase Proteins
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The AcNPV-Chi, CpGV-Chi, and S. marcescens-ChiC proteins were overexpressed as fusion proteins with the 6×His-tag at their N-terminal in E. coli BL21 (DE3) cells. Chitinase proteins were purified by using the MagneHis™ Protein Purification System Kit (Promega) and dialyzed for 24 h through 1 l of 1X PBS buffer, pH 7.5. The identification and purity of the samples were confirmed by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and subsequently Coomassie staining. Western blot analysis was also performed to demonstrate heterologous gene expression at the immunological level. Electrophoresed proteins were transferred to the nitrocellulose membrane. Immunedetection was performed using 1:1000 diluted polyclonal rabbit anti-His-taq antibodies (Abcam) and subsequently 1:1000 diluted polyclonal alkaline phosphataseconjugated goat-anti-rabbit IgG (Millipore). The binding of the antibodies was then visualized with the NBT-BCIP substrate system (Roche).
3
Western Blotting of CTCF and Rad21
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CEF, erythrocytes and HEK293 protein extracts, (52 mcg, corresponding to 110 000 cells) along with the protein marker SM0661, Fermentas, were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to 0.45 mkm Polyvinylidene difluoride membrane for Western blotting (Biotrans, US) using a mini transfer cell (Bio-Rad, Hercules CA). After transfer the membrane was blocked by an 1 h incubation in 5% fat-free dry milk dissolved in PBST (1× PBS, 0.1% Tween-20), washed three times in PBST and incubated with primary antibody anti-CTCF (Cell Signaling Technology, 3418S) or with primary antibody anti-Rad21 (Abcam, ab16-473-100) (3 h, RT). After three washes in PBST the membrane was incubated with affinity purified alkaline phosphatase-conjugated goat anti-rabbit IgG (A3812, Sigma) or with affinity purified alkaline phosphatase-conjugated goat anti-mouse IgG (A3562, Sigma). Three washes in PBST of the membrane was followed by 5 min incubation in the buffer for alkaline phosphatase (100 mM Tris–HCl, pH 9.5, 100 mM NaCl, 10 mM MgCl2) and the bound antibodies were visualized using nitro-blue tetrazolium/5-bromo-4-chloro-3′-indolyphosphate (BCIP/NBT) substrate for alkaline phosphatase (Sigma).
The protein marker was visualized by amido black staining according to a standard protocol.
The protein marker was visualized by amido black staining according to a standard protocol.
4
Immunoblotting of Microsomal Proteins
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Microsomal fractions were isolated as previously described (Jasiński et al., 2001 (link)) from 150 mg of Medicago hairy roots or 300 mg of BY2 cells. The proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore) by electroblotting (semi-dry; apparatus; Bio-Rad). The membrane was incubated with a primary polyclonal antibody specific for MtABCG10 (Banasiak et al., 2013 (link)) or with a primary antibody against H+-ATPase (W1G) (Morsomme et al., 1998 (link)). The secondary antibody was an alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma).
5
Quantification of Anti-F1 Antibodies
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Serum proteins (10 μl) were separated by 4–12% gradient SDS-PAGE (Invitrogen) and were stained using a Silver Stain Plus kit (Bio-Rad). For Western blot analysis, electrophoresed samples were transferred to nitrocellulose membranes (Invitrogen). Membranes were developed with polyclonal antibodies against hemopexin (Abcam ab 90947), transferrin (Abcam ab 82411) and β-actin (Sigma A5060), followed by HRP-conjugated goat anti-rabbit IgG. Anti-F1 ELISA in the serum of immunized mice was performed as described previously (Levy et al., 2011 (link), 2016 (link)). Briefly, microtiter plates were coated with 500 ng of purified rF1 [provided by the Biotechnology Department at IIBR, produced as described in Holtzman et al. (2006 (link))]. Tested sera were serially diluted in 2-fold dilutions in a final volume of 50 μl and were incubated in the wells for 1 h at 37°C. Alkaline phosphatase-conjugated goat anti-rabbit IgG (1/2,000 dilution, Sigma) was used as the 2nd layer for rabbit anti-F1 IgG titer determination. Titers were defined as the reciprocal values of the endpoint serum dilutions that displayed OD405 values 2-fold higher than the normal serum controls.
6
Western Blot Analysis of ATX3
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Protein was extracted with TRIzol reagent (Invitrogen, USA). Samples were run on discontinuous 10% SDS-polyacrylamide gels (PAGE) and both the stacking and separating portions of the gel were blotted onto Hybond-C Extra nitrocellulose membranes (GE Healthcare, USA). Blots were incubated with affinity-purified ATX3 antiserum (1:1,000, kindly provided by Dr. H. Paulson), followed by alkaline phosphatase-conjugated goat anti-rabbit IgG (1:20,000, Sigma, USA).
Aggregates are insoluble in SDS-PAGE and, therefore, remain in the stacking portion of the gel. ATX3 monomer/aggregate ratios were calculated based on band densitometry using ImageJ software.
Aggregates are insoluble in SDS-PAGE and, therefore, remain in the stacking portion of the gel. ATX3 monomer/aggregate ratios were calculated based on band densitometry using ImageJ software.
7
Protein Extraction and Western Blot Analysis
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Proteins were extracted from frozen fruit samples in extraction buffer (2% SDS, 60 mM DTT, 20% glycerol, 40 mM Tris-HCl ph 8.8). After 8 min boiling, samples were centrifuged at 7,500 xg for 15 minutes and then rinsed for 1 h in 3 volumes acetone and 20 mM DTT. After dehydration, pellet was resuspendend in 1% SDS. SDS-PAGE was performed as described by Laemmli [31 (link)]. After gel electrophoresis, proteins were blotted onto a 0.45 μm nitrocellulose membrane by a Trans-Blot cell (Bio-Rad, Milano, Italy) containing transfer buffer (25 mM Tris, 192 mM glycine and 20% (v/v) methanol, pH 8.3). Membranes were blocked with 5% (w/v) non-fat dry milk powder in TBS-T (20 mM Tris, 150 mM NaCl and 0.05% (v/v) Tween 20, pH 7.5) for 1 h and then incubated with anti-endo-PG (1 μg/mL) and anti-CHS (0.4 μg/mL) for 1h in TBS-T. After three rinses in TBS-T for 5 min each, membranes were soaked for 1 h with alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma-Aldrich) diluted 1:15000 in TBS-T. Membranes were then washed three more times with TBS-T. Immunoreactive bands were detected by Sigma Fast BCIP/NBT as an alkaline phosphatase substrate according to the manufacturer’s protocol.
8
Lipid Signaling Molecules Protocol
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FB1, sphinganine, phytosphingosine, and N-Hexanoyl-D-Erythro-sphingosine (C6:0 ceramide), were purchased from Sigma-Aldrich (St. Louis, MO, USA) and N-Palmitoyl-D-Erythro-sphingosine (C16:0 ceramide) from ICN (Irvine, CA, USA). Optiprep and 1,6 diphenyl-1,3,5-hexatriene (DPH) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Triton X-100 was purchased from Pierce (Rockford, Ill). Antibody against beet PM H+-ATPase was a kind gift from Dr. Luis E. González de la Vara (CINVESTAV, Irapuato, Mexico). Antibody against 14-3-3 β isoform was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Alkaline-phosphatase conjugated goat anti-rabbit IgG was obtained from Sigma-Aldrich (St. Louis, MO, USA). Uridine diphosphate glucose, [glucose-1-3H] was purchased from NEN Life Sciences Products, Inc. (Boston, MA, USA). All the other chemicals were of the highest purity available.
9
Detecting PVX Accumulation in Plants
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The accumulation of wild type and mutant PVX in N. benthamiana and tomato plants were determined using Western blotting. Total proteins were extracted from the systemic leaves of N. benthamiana and tomato plants, separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to a nitrocellulose (NC) membrane. The NC membrane was blocked with 5% defatted milk powder in pH 7.6 Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h, incubated in antiserum against PVX CP diluted at 1:1000 (V/V) for 1 h, followed by 1 h incubation with alkaline phosphatase conjugated goat anti-rabbit IgG diluted in 1:50000 (Sigma) and finally colorized with NBT and BCIP.
10
Analysis of Viral RNA and Protein
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Samples were harvested and processed for the analysis of viral RNA and protein as previously described (Weiland and Edwards, 1994 (link)). Total protein (for Western blots) and nucleic acids (for Northern blots) from 105 protoplasts were separated on 12% sodium dodecylsulfate polyacrylamide gels and 1% denaturing agarose gels, respectively, as indicated (Edwards and Weiland, 2010 (link)). Proteins were electroblotted from polyacrylamide gels to nitrocellulose membranes (Schleicher and Schuell [Keene, N.H. USA], BA-85, 0.2 µ), which subsequently were incubated in a 1:1000 dilution of rabbit anti-OBDV, followed by incubation in a 1:2000 dilution of alkaline phosphatase-conjugated goat anti-rabbit IgG (Product #A0418, Sigma-Aldrich, St. Louis, MO USA). Protein complexes were detected on a Kodak Image Station 2000 MM following treatment of the blot with LumiPhos WB (Thermo Scientific, Lafayette, CO USA). Nucleic acids transferred to positively-charged nylon membranes by capillary blotting (Roche Applied Science, Indianapolis, IN USA) were probed with a digoxigenin-labeled, denatured dsDNA probe representing nucleotides 5929–6508 of OBDV-2r. After incubating blots with alkaline phosphatase-conjugated anti-digoxigenin IgG, CDP-Star (Roche Applied Science, Indianapolis, IN USA) was added and viral RNAs were detected by chemiluminescence.
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