Binding of C3 to bacterial cells was determined by ELISA as previously described (Qadi et al., 2017 (link)). Bacterial cells (1x109 CFU) were washed with PBS and incubated for 15 min at 37°C with human serum (20%) supplemented or not with FHR-1 (20 µg/ml). HI-serum was used as control. Next, bacteria were washed and incubated for 2 h at 37°C in 50 mM carbonate-bicarbonate buffer (pH 9.0) containing 1 M NH4OH to disrupt ester bonds between C3 fragments and the bacterial surface. Serial dilutions of the eluted cell-bound C3 were used to coat microtiter plate wells at 4°C for 18 h. Wells were blocked with PBS-BSA, incubated sequentially with a rabbit polyclonal anti-human C3 (Abcam, ab117244) which detects C3, C3b and iC3b, and alkaline phosphatase-labeled goat anti-rabbit immunoglobulin G (Sigma), and developed with p-nitrophenyl phosphate (Sigma) in 50 mM carbonate-bicarbonate buffer (pH 9.6) plus 5 mM MgCl2. Controls values were < 0.1 optical density units and were subtracted from the experimental values.
Goat anti rabbit immunoglobulin g
Manufactured by Merck Group
Goat anti-rabbit immunoglobulin G is a laboratory reagent used as a secondary antibody in various immunological techniques. It is produced by immunizing goats with rabbit immunoglobulin G (IgG) and then purifying the resulting antibodies. The primary function of this product is to bind to and detect rabbit primary antibodies, allowing for the identification and quantification of target analytes in samples.
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3 protocols using goat anti rabbit immunoglobulin g
1
Quantifying C3 Binding to Bacteria
2
Protein Fractionation and Western Blot Analysis
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Protein samples from the fractionation experiments (total, cytoplasmic, and nuclear) were resuspended in sample loading buffer and heated up at 65°C for 10 min before loading in a 10–20% polyacrylamide gradient gel (Bio-Rad) and transferred to polyvinylidene fluoride membranes. Membranes were probed with anti-H3 antibody (Abcam 1791) or anti-alcohol dehydrogenase (Agrisera AS10 685) at a dilution of 1:5,000 and the secondary antibody used was goat anti-rabbit immunoglobulin G coupled to unmodified horseradish peroxidase (Sigma) at a 1:10,000 dilution. Detection was done using the ECL Western Blotting Detection Reagent (Amersham) and signal detected with Image Quant LAS4000 (GE).
3
Immunoblot Analysis of Protein Expression
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The immunoblot assay used to detect gene expression at the protein level was performed following a method described previously (Choi et al. 2008) . Briefly, total proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and were transferred into polyvinylidene fluoride membranes. The membranes were incubated with primary anti-Flag antibody at a 1:5,000 dilution. Goat antirabbit immunoglobulin G (Sigma-Aldrich) with horseradish peroxidase conjugate was used as a secondary antibody, diluted at 1:20,000.
To further investigate the protein expression of CaCBL1 in N. benthamiana cells, total proteins were extracted from N. benthamiana leaves infiltrated with Agrobacterium sp. strain GV3101 carrying 35S:CaCBL1-GFP or 35S:GFP constructs, as described previously (Choi et al. 2008) . The proteins of nuclear fractionation and cytoplasm fractionation were extracted using a plant nuclear or cytoplasmic protein extraction kit (BestBio), respectively. The total nuclear or cytoplasmic proteins were subjected to immunoblot analysis. Anti-H3 (Proteintech) and antiheat-shock complex 70 (Proteintech) were used as nuclear and cytosolic protein markers, respectively.
To further investigate the protein expression of CaCBL1 in N. benthamiana cells, total proteins were extracted from N. benthamiana leaves infiltrated with Agrobacterium sp. strain GV3101 carrying 35S:CaCBL1-GFP or 35S:GFP constructs, as described previously (Choi et al. 2008) . The proteins of nuclear fractionation and cytoplasm fractionation were extracted using a plant nuclear or cytoplasmic protein extraction kit (BestBio), respectively. The total nuclear or cytoplasmic proteins were subjected to immunoblot analysis. Anti-H3 (Proteintech) and antiheat-shock complex 70 (Proteintech) were used as nuclear and cytosolic protein markers, respectively.
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