Soluble Gal1 was determined using an in-house Enzyme-Linked Immunosorbent Assay (ELISA) as described (34 (link)). In brief, high-binding 96-well microplates (Costar; Corning) were coated with capture Ab (2 μg/mL purified rabbit anti-human Gal1 polyclonal IgG) in 0.1 M sodium carbonate, pH 9.5. After incubation for 18 h at 4 °C, wells were rinsed three times with wash buffer (0.05% Tween-20 in PBS) and incubated for 1 h at room temperature with blocking solution (2% Bovine Serun Albumin (BSA) in Phosphate buffer saline (PBS)). Samples and standards (100 μL) were diluted in 1% BSA–Tween-20 and incubated for 18 h at 4 °C. Plates were then washed and incubated with 100 ng/mL biotinylated detection Ab (purified rabbit anti-human Gal1 polyclonal IgG) for 1 h. Plates were rinsed three times before adding 0.33 μg/mL HRP-labeled streptavidin (Sigma-Aldrich) for 30 min. After washing, 100 μL 3,3’,5,5’-Tetramethylbenzidine (TMB) solution (0.1 mg/mL tetramethylbenzidine and 0.06% H2O2 in citrate–phosphate buffer, pH 5.0) was added to plates. The reaction was stopped by adding 2N H2SO4. Optical densities were determined at 450 nm in a Multiskan MS Microplate Reader (Thermo Fisher Scientific). A standard curve ranging from 2.5 to 160 ng/mL human rGal1 was run in parallel.
Hrp labeled streptavidin
Manufactured by Merck Group
Sourced in United States
HRP-labeled streptavidin is a laboratory reagent used in various immunoassay techniques. Streptavidin, a protein derived from the bacteria Streptomyces, has a high affinity for biotin, a small molecule commonly used to label target analytes. The HRP (Horseradish Peroxidase) label attached to the streptavidin molecule enables the detection and visualization of biotinylated targets through colorimetric or chemiluminescent signals.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse kappa polyclonal antibody conjugated with biotin, by Bio-Rad (2 mentions)
96 well high binding microplates, by Corning (1 mentions)
Multiskan ms microplate reader, by Thermo Fisher Scientific (1 mentions)
Gen5 2, by Agilent Technologies (1 mentions)
Synergy h1, by Agilent Technologies (1 mentions)
Bca assay, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Mem medium, by Thermo Fisher Scientific (1 mentions)
Guanidine hydrochloride, by Merck Group (1 mentions)
Iodoacetamide iam, by Merck Group (1 mentions)
Ammonium carbonate, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Omalizumab, by Novartis (1 mentions)
Poly l lys, by Merck Group (1 mentions)
5 protocols using hrp labeled streptavidin
1
Quantifying Soluble Galectin-1 by ELISA
2
Quantifying Antibody Concentrations in Cell Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibody concentrations in cell culture media were measured using a standard sandwich ELISA on plates coated with sheep anti-mouse Fab polyclonal antibody (Jackson Laboratory, cat. #515-005-072, lot #105461). Goat anti-mouse kappa polyclonal antibody (1:3,000, BioRad, cat. #105008, batch #160617), HRP-labeled streptavidin (1:40,000, Sigma), and the TMB substrate for HRP (BD Bioscience) were used for detection. The HRP-dependent colorogenic reaction was stopped with 1 M HCl, and the absorbance at 450 nm was read using the microplate spectrophotometer Synergy H1 operated by Gen5 2.00 Software (BioTek). Purified M18 (IgG3) and MCP21 (IgG1, Sigma) were used as standards. Concentrations of muteins were calculated based on their Fab type (IgG1- or IgG3-type Fab). BCA assay (Sigma) was used for measurements of purified antibody concentrations with bovine γ-globulin (Thermo) as a reference.
3
Comparative Analysis of Omalizumab and Biosimilar
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Omalizumab was purchased from Novartis (Basle, Switzerland). L0H0 was an in-house produced Omalizumab biosimilar antibody. MEM medium, FBS, Formic acid and acetonitrile were obtained from Thermo Fisher Scientific (Pittsburg, PA, USA). Iodoacetamide (IAM), dithiothreitol (DTT), urea, β-mercaptoethanol, HRP-labeled streptavidin, guanidine hydrochloride and ammonium carbonate were obtained from Sigma-Aldrich (St. Louis, MO, USA).
4
Western Blot Analysis of Mouse Ig Subclasses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were resolved in 8 or 12% polyacrylamide gels under non-reducing or reducing conditions according to the protocol of Laemmli (14 (link)). After wet electrotransfer onto PVDF membrane and blocking with 4% skim milk in PBS, the samples were probed with anti-mouse kappa polyclonal antibody conjugated with biotin (1:3,000, BioRad) and HRP-labeled streptavidin (1:40,000, Sigma). Alternatively, rabbit anti-HA tag polyclonal antibody (1:10,000, Abcam, cat. #ab9110) and HRP-labeled goat anti-rabbit F(ab’)2 polyclonal antibody (1:10,000, Sigma, cat. #A6667, lot #SLBG3029) were used. Mouse IgG3 heavy chain was detected using goat antiserum to mouse IgG3 (1:500, Sigma, cat. #ISO2) and rabbit anti-goat polyclonal antibody (1:5,000, Sigma, cat. #A4174). Bands were visualized using Immobilon Western Chemiluminescent Substrate for HRP (Millipore). The images were captured and analyzed using Fusion Fx apparatus with the Fusion Capt Advance Fx5 program (Vilbert Lourmat, France).
5
Quantifying Cell-Bound Antibodies via Colorimetric Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Polystyrene plates were coated with 50 µg/ml poly-l -Lys (Sigma) for 1 h at room temperature (RT). Then, 100 µl of 0.1% (hematocrit) suspension of red blood cells in PBS were added to the wells and the cells were allowed to settle for 1 h at RT. After gentle aspiration of the solution, the cells were fixed using 0.025% glutaraldehyde for 40 min. Endogenous peroxidase activity was blocked with 3% H2O2 for 1 h. The plates were blocked overnight with 0.2% gelatin in PBS containing 0.05% Tween-20 at 4°C. Then, the cells were incubated with serial dilutions of cell culture media containing analyzed antibodies followed by detection with anti-mouse kappa polyclonal antibody conjugated with biotin (1:3,000, BioRad) and HRP-labeled streptavidin (1:40,000, Sigma). All reagents were diluted in the blocking buffer. The colorogenic reaction was performed, and the absorbance at 450 nm was measured 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!