Endotoxin-free ovalbumin (EndoGrade Ovalbumin, Hyglos GmbH) was opsonised with a polyclonal rabbit anti-ovalbumin antibody (Sigma-Aldrich, St. Louis, MO) at 37 °C for 1 hour.
Rabbit anti ovalbumin antibody
Manufactured by Merck Group
Rabbit anti-ovalbumin antibody is a laboratory reagent used for the detection and quantification of ovalbumin, a protein found in egg white. This antibody is produced by immunizing rabbits with ovalbumin and can be used in various immunological techniques, such as ELISA, Western blotting, and immunohistochemistry, to identify and measure the presence of ovalbumin in samples.
Automatically generated - may contain errors
Lab products found in correlation
Ovalbumin (ova), by Thermo Fisher Scientific (2 mentions)
Goat anti c4 antibody, by Complement Technology (2 mentions)
Donkey anti goat igg hrp antibody, by Santa Cruz Biotechnology (2 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
2 n morpholino ethanesulfonic acid mes buffer, by Merck Group (1 mentions)
Dq ovalbumin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 conjugated ovalbumin, by Thermo Fisher Scientific (1 mentions)
C6534, by Merck Group (1 mentions)
O34784, by Thermo Fisher Scientific (1 mentions)
7 protocols using rabbit anti ovalbumin antibody
1
Antibody-Opsonized Ovalbumin Preparation
2
Endocytosis Quantification via Bead Uptake
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Endocytosis was tested using 1 μm fluorescent yellow-green carboxylate-modified polystyrene latex beads (Sigma-Aldrich) coated with ovalbumin. Briefly, after being washed in MES (2-(N-morpholino)ethanesulfonic acid) buffer (pH = 6.1) (Sigma-Aldrich), the beads were suspended to give a 1% suspension and incubated with 4 mg/ml ovalbumin (Sigma-Aldrich) in MES overnight with gentle shaking following the manufacturer’s instructions. Thereafter, the beads were washed in PBS and stored as a 1% suspension. Cells pre-treated with bacterial lysates were incubated with bacterial lysates for 90 min in serum-free medium before adding beads, dissolved at 1:5000 in the medium. Cells were fixed 60 min later with 4% formalin and stained with rabbit anti-ovalbumin antibody (1:500 dilution; Sigma-Aldrich) without membrane permeabilization (thus staining only extracellular beads) and goat anti-rabbit Cy3 secondary antibody as described above. Phagocytosed beads showed green fluorescence (their native fluorescence), but were anti-ovalbumin-negative. Extracellular beads were both fluorescently green and red (Cy3 ovalbumin staining).
3
Ovalbumin Immune Complex Stimulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Endotoxin-free ovalbumin (1 mg/mL, Hyglos) was opsonized with a polyclonal rabbit anti-ovalbumin antibody (3.7 mg/mL, Sigma-Aldrich) at 37°C for 1 hour, at a 1:10 (v/v) ratio, to form insoluble immune complexes (IC), for stimulation of cells in culture or administration in vivo. Cells were harvested at defined time points and washed to remove free ICs. For RNA sequencing experiments, BMDMs and BMDCs were stimulated with immune complexes of ovalbumin for 4 hours. For phagocytosis assays, Alexa Fluor (AF)647-conjugated ovalbumin was used (Thermo Fisher), and conjugated with antibody as described above. For in vivo phagocytosis, 0.33 g/kg AF647-conjugated ovalbumin was opsonized with 3.2 g/kg polyclonal rabbit anti-ovalbumin antibody before injection. Phagocytic degradation products were identified by substituting Ova-647 with DQ™ Ovalbumin (Invitrogen), a conjugate that exhibits fluorescence only after proteolytic degradation.
4
Antibody-Opsonized Ovalbumin Preparation
5
Immune Complex Formation and Phagocytosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For ovalbumin immune complexes, endotoxin-free ovalbumin (no. 321000; Hyglos) was opsonized with a polyclonal rabbit anti-ovalbumin antibody (C6534, Sigma; 1:140, wt/wt) at 37 °C for 1 h. For in vitro phagocytosis assays, Alexa Fluor 647-conjugated ovalbumin was used (O34784; Thermo Fisher). For IgG immune complexes, human IgG (5172-9017; AbD Serotec)/mouse IgG (ab36355; Abcam) was opsonized with monoclonal human anti-human IgG antibody (HCA059; AbD Serotec)/goat F(ab′)2 anti-mouse IgG-(Fab′)2 antibody (ab98754; Abcam; 1:200, wt/wt) at 37 °C for 1 h. For in vivo experiments, 0.33 g/kg Alexa Fluor 647-conjugated ovalbumin (O34784; Thermo Fisher) was opsonized with 3.2 g/kg polyclonal rabbit anti-ovalbumin antibody (Sigma-Aldrich) at 37 °C for 1 h before injection. Details of systemic lupus erythematosus immune complexes are provided in the SI Appendix .
6
C4 Activation Assay for Ovalbumin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 4
The C4 activation assay was adapted from Mallik et al., 2005. J. Med. Chem. 48, 274-286. Wells of Immulon-2, 96 well plates were coated with 50 μl of 1.0 mg/ml ovalbumin (Fisher) in coating buffer and incubated overnight at 4° C. The plates were washed with PBS/T and blocked with 3% BSA/PBS for 2 hours at room temperature. The plates were washed again and then incubated with a rabbit anti-ovalbumin antibody (Millipore) diluted in 3% BSA/PBS at 1:2,000 for 1 hour at room temperature. During this incubation, the peptides were diluted to 0.5 mM in 10% NHS/GVBS++ and incubated for 15 minutes at 37° C. The plates were then washed, and the pre-incubated samples were added to the plates at a 1:4 dilution in GVBS++ and incubated for 30 minutes at room temperature. Afterwards, the plates were washed and goat anti-C4 antibody (Complement Technologies, Inc.) was added at a dilution of 1:2,000 in 3% BSA/PBS for 1 hour, followed by another wash and a donkey anti-goat IgG-HRP antibody (Santa Cruz Biotechnology, Inc.) diluted to 1:2,000 in 3% BSA/PBS for 1 hour. The plates were then developed and absorbance values determined as described above.
7
C4 Activation Assay for Ovalbumin Antibody
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 4
The C4 activation assay was adapted from Mallik et al., 2005. J. Med. Chem. 48, 274-286. Wells of Immulon-2, 96 well plates were coated with 50 μl of 1.0 mg/ml ovalbumin (Fisher) in coating buffer and incubated overnight at 4° C. The plates were washed with PBS/T and blocked with 3% BSA/PBS for 2 hours at room temperature. The plates were washed again and then incubated with a rabbit anti-ovalbumin antibody (Millipore) diluted in 3% BSA/PBS at 1:2,000 for 1 hour at room temperature. During this incubation, the peptides were diluted to 0.5 mM in 10% NHS/GVBS++ and incubated for 15 minutes at 37° C. The plates were then washed, and the pre-incubated samples were added to the plates at a 1:4 dilution in GVBS++ and incubated for 30 minutes at room temperature. Afterwards, the plates were washed and goat anti-C4 antibody (Complement Technologies, Inc.) was added at a dilution of 1:2,000 in 3% BSA/PBS for 1 hour, followed by another wash and a donkey anti-goat IgG-HRP antibody (Santa Cruz Biotechnology, Inc.) diluted to 1:2,000 in 3% BSA/PBS for 1 hour. The plates were then developed and absorbance values determined as described above.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!