Journal: Poultry Science

Article Title: A novel and quick egg yolk immunoglobulin y antibody extraction method leveraging the protein liquid-liquid phase separation principle

doi: 10.1016/j.psj.2025.104804

Figure Lengend Snippet: PEG 8000 induced LLPS and purification of egg yolk IgY. Notes: After the addition of PEG, condensates could be observed under a light microscope ( A-H ). Scale bar = 5 μm. (I) ELISA titration curve comparing the antigen biding properties of IgY extracted with various concentrations of PEG. The coating antigen for the ELISA was whole cells of Staphylococcus epidermidis . Serial dilutions of the IgY (1000 – 10 μg/mL) were prepared and used as the primary antibodies and detected using HRP-conjugated rabbit anti-chicken IgY secondary antibody. ( J ) 12 % SDS-PAGE analysis of proteins obtained after second PEG 8000 addition after delipidation and as well as PEG 6000 precipitation. M: Prestained molecular weight marker (Shanghai Shengen Biotech.). The bands corresponding to the heavy chain (65-70 kDa) and light chain (25 kDa) of IgY are marked. ( K ) Western blot analysis of IgY purified using PEG 8000. Purified IgY separated on an SDS-PAGE gel was transferred onto nitrocellulose membrane, probeb with rabbit anti-chicken IgG HRP conjugate and visualized using 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate.

Article Snippet: The proteins on the gel were transferred onto nitrocellulose membrane (Immobilon-NC, Solarbio, China) for Western blotting using a BioRad Mini Trans-blot blotter (Bio Rad, Hercules, CA, USA) set at 100 V for 2 h. After blotting, the membranes were blocked with a 5 % bovine serum albumin (BSA) in phosphate buffer saline containing 0.05 % (v/v) Tween-20 (PBST) at 4 °C for 16 h. The blocking solution was discarded and the membrane washed three times using PBST for 5 min each.

Techniques: Purification, Light Microscopy, Enzyme-linked Immunosorbent Assay, Titration, SDS Page, Molecular Weight, Marker, Western Blot, Membrane

Journal: Poultry Science

Article Title: A novel and quick egg yolk immunoglobulin y antibody extraction method leveraging the protein liquid-liquid phase separation principle

doi: 10.1016/j.psj.2025.104804

Figure Lengend Snippet: Affinity of IgY isolated using novel PEG 8000 method. Notes: The affinity of the IgY to antigens was assessed using Western blot analysis. Lysates of Staphylococcus epidermidis were separate on 12 % reducing SDS-PAGE gels and stained ( A ) or transferred onto nitrocellulose membranes. The IgY isolated using the PEG 8000 ( B ) or PEG 6000 ( C ) methods were used as primary antibodies. The blots were visualised using Chemiluminiscence subtrate (Beyotime, Inc, China). (D) Titration ELISA using the extracted IgY using the novel PEG 8000 method compared with the commonly used PEG 6000 purification method. The experiment was repeated three times and the EC 50 values calculated from the non-linear regression of the curves were compared using Whitney-Mann U test. *The difference between the EC 50 values for the two isolation methods was not Statistically significant ( p > 0.05).

Article Snippet: The proteins on the gel were transferred onto nitrocellulose membrane (Immobilon-NC, Solarbio, China) for Western blotting using a BioRad Mini Trans-blot blotter (Bio Rad, Hercules, CA, USA) set at 100 V for 2 h. After blotting, the membranes were blocked with a 5 % bovine serum albumin (BSA) in phosphate buffer saline containing 0.05 % (v/v) Tween-20 (PBST) at 4 °C for 16 h. The blocking solution was discarded and the membrane washed three times using PBST for 5 min each.

Techniques: Isolation, Western Blot, SDS Page, Staining, Titration, Enzyme-linked Immunosorbent Assay, Purification