Affinity of the purified Nanobodies (Nb94 and Nb60) was determined using ELISA method. TIM-3 antigen (HAVCR2 Protein /Sino Biological Inc.) was coated in a 96 well microplate wells (Nunc, Denmark) in different concentrations (5 and 10 μg/ml). After washing and blocking, the Nb was added at 0.001, 0.01, 0.1, 1, 10 nM concentrations. HRP-conjugated anti-His antibody (Abcam, UK) was added and the immune reactivity was assessed with TMB substrate. The reaction was stopped with a stop solution and OD was measured at 450 nm using a microplate reader (Hyperion).
Hrp conjugated anti his antibody
Manufactured by Abcam
Sourced in United Kingdom
HRP-conjugated anti-His antibody is a labelled antibody that specifically binds to the histidine (His) tag. It is commonly used in various immunoassays and detection methods to identify and visualize proteins containing a His tag.
Automatically generated - may contain errors
3 protocols using hrp conjugated anti his antibody
1
Affinity Profiling of Anti-TIM-3 Nanobodies
2
ELISA Analysis of RBM-CH3(scaffold) Binding to ACE2
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The ELISA was performed to characterize the binding of RBM-CH3(scaffold) to ACE2. Maxisorp plates (Nunc) were coated with 100 μl of ACE2-Fc protein (2 μg/ml) or BSA as control in 1× carbonate/bicarbonate buffer, pH 9.6 for overnight at 4 °C. Next day, the plate was blocked by 250 μl of PBS containing 5% skimmed milk. Different concentration of RBM-CH3(scaffold) protein ranging from 16 μg/ml to 0.25 μg/ml in ½ fold serial dilution was added to the wells (100 μl/well) and incubated for 2 h at RT. The wells were then washed and HRP-conjugated anti-His antibody (Abcam) in 1:2000 dilution were used for developing ELISA.
For characterizing the binding of immunized sera with the RBM-CH3(scaffold), the RBD and the prefusion spike protein, Maxisorp plates (Nunc) were coated with 100 μl of the proteins at concentration of 2 μg/ml in 1× coating buffer for overnight at 4 °C. Next day, the plates were blocked using 250 μl of PBS containing 5% skimmed milk. The immunized sera were diluted serially 1:3 times in the dilution buffer with a starting concentration of 1:300 dilution and incubated for 2 h followed by washing and blocking. HRP-conjugated anti mouse secondary antibody (Jackson Immuno Research, USA) in 1:2000 dilution was used for developing ELISA.
For characterizing the binding of immunized sera with the RBM-CH3(scaffold), the RBD and the prefusion spike protein, Maxisorp plates (Nunc) were coated with 100 μl of the proteins at concentration of 2 μg/ml in 1× coating buffer for overnight at 4 °C. Next day, the plates were blocked using 250 μl of PBS containing 5% skimmed milk. The immunized sera were diluted serially 1:3 times in the dilution buffer with a starting concentration of 1:300 dilution and incubated for 2 h followed by washing and blocking. HRP-conjugated anti mouse secondary antibody (Jackson Immuno Research, USA) in 1:2000 dilution was used for developing ELISA.
3
Quantifying Fv2 Protein Binding to Gcn4 and JunLZ
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Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the binding of purified Fv2 proteins to Gcn4 and JunLZ. ELISA plates were coated overnight at 4°C with 50 μL/well of each antigen in PBS (10 μg/mL). Plates were then blocked at room temperature for 2 h with 2% nonfat milk in PBS. After washing plates with PBS supplemented with 0.1% Tween 20 (PBST), purified protein samples serially diluted in PBS with 50 μg/mL BSA (PBS-BSA) were added to the plates (50 μL/well). Plates were incubated for 1 h at room temperature and then washed with PBST. HRP-conjugated anti-His antibody (1:10,000; Abcam) in PBS-BSA was added to the plates (50 μL/well). After 1 h of incubation at room temperature, plates were washed and then incubated with SigmaFast OPD HRP substrate (Sigma) for 20 min. The reaction was quenched with 3N H2SO4, and the absorbance of the wells was measured at 490 nm.
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