Journal: Biotechnology and Bioengineering

Article Title: Transferrin Purification, Biophysical Characterization, and Lung Biodistribution in Sickle Cell Disease Mice

doi: 10.1002/bit.70012

Figure Lengend Snippet: Trypsin digest mass spectrometry analysis of the protein cocktail and purified Tf. Both figures show the ten most abundant proteins and cover > 99% of all proteins in the sample. (A) Represents the protein composition of the protein cocktail that was used as the starting material for the purification of Tf. The error bars were derived from three separate purifications. (B) Represents the protein composition of the purified Tf. The error bars were derived from three separate purifications. Abbreviations: ALB, albumin; APOA1, apolipoprotein A‐I; APOA2, apolipoprotein A‐II; APOH, beta‐2‐glycoprotein; CFI, complement factor I; CP, ceruloplasmin; GC, vitamin D‐binding protein; HBA1, hemoglobin subunit alpha; HBB, hemoglobin subunit beta; HP, haptoglobin; HPR, haptoglobin related protein; HPX, hemopexin; IGG1, immunoglobulin gamma‐1 heavy chain; IGHG4, immunoglobulin heavy constant gamma 4; RBP4, retinol‐binding protein 4; SERPINA1, alpha‐1‐antitrypsin; TF, transferrin.

Article Snippet: Auto‐oxidation of human hemoglobin (Hb) was monitored on a Hewlett‐Packard 8452A spectrophotometer (HP, Palo Alto, CA).

Techniques: Mass Spectrometry, Purification, Derivative Assay, Binding Assay

Journal: Scientific Reports

Article Title: Biochemical and physiological characterization of Aedes aegypti midgut chymotrypsin

doi: 10.1038/s41598-025-93413-7

Figure Lengend Snippet: Protein gel electrophoresis analysis of AaCHYMO, human and bovine chymotrypsin digestion assays with the three major human blood proteins: human serum albumin (HSA) ( A ), hemoglobin (Hb) ( B ), and immunoglobulin G (IgG) ( C ), incubated for a total of 60 min post-protease addition. Time = 0 min is the no protease control in each reaction. ( A ) Comparing the HSA digestion fragments of AaCHYMO only one single cleavage product is observed (teal arrow), as opposed to several digestion fragments observed in human and bovine chymotrypsin digestion assays. (B ) In the Hb digestion assays, only one single cleavage product is observed (red arrow), while the higher molecular weight (MW) Hb band protein species (blue arrows) stays intact throughout the whole reaction time but is readily degraded by human and bovine chymotrypsin. ( C ) In the IgG digestion assays, the two top bands in between the 50 and 70 kDa MW marker (red and orange arrows) remain intact throughout the reaction time, with only a single cleavage product observed appearing at 20 min after protease addition (aqua arrow). Human and bovine chymotrypsin readily degrade the two bands between the 50 and 70 kDa MW marker (red and orange arrows in the AaCHYMO digestion reaction), with more observable cleavage products below the 30 kDa MW marker observed. The protein gels shown in the figure were cropped for clarity. The original unaltered protein gels are presented in Supplementary File 1.

Article Snippet: AaCHYMO was incubated with human serum albumin (HSA) (Abcam #AB205808), hemoglobin (Hb) (MP Biomedicals, LLC.

Techniques: Nucleic Acid Electrophoresis, Incubation, Control, Molecular Weight, Marker

Journal: Scientific Reports

Article Title: Biochemical and physiological characterization of Aedes aegypti midgut chymotrypsin

doi: 10.1038/s41598-025-93413-7

Figure Lengend Snippet: AaCHYMO is predicted to interact with IgG and less likely to interact with human albumin or hemoglobin. Modeled structure of AaCHYMO by AlphaFold2 (gray) was submitted to HDOCK to predict potential interactions with ( A ) human serum albumin (PDB ID: 1E7H, slate blue), ( B ) human hemoglobin (PDB ID: 1GZX, salmon), and ( C ) human neutralizing IgG (PDB ID: 1HZH, cyan). Predictions suggest the latter’s potential to interact and have residues close to the catalytic site of the enzyme (~ 7Å). Results suggest that the two other human proteins can potentially engage, but inconclusive for full cleavage to occur. Predicted interactions are shown as black dashed lines, while measured distances to the catalytic serine are shown as yellow dashed lines. Heme groups are displayed as sticks (brown). Catalytic residues are displayed as sticks (gray) and labeled (H48, D97, and S193). Structures are represented by cartoons and a transparent surface. All images were generated with PyMOL software (v2.5.7).

Article Snippet: AaCHYMO was incubated with human serum albumin (HSA) (Abcam #AB205808), hemoglobin (Hb) (MP Biomedicals, LLC.

Techniques: Labeling, Generated, Software

Journal: Blood Advances

Article Title: Fc mutagenesis enhances the functionality of anti-RhD monoclonal antibodies

doi: 10.1182/bloodadvances.2024015082

Figure Lengend Snippet: Fc modification enhances RBC clearance through ADCC. (A-B) ADCC assays were performed using monocyte-depleted PBMCs in the presence bromelain-treated (A) or untreated (B) RhD pos RBCs opsonized with anti-RhD Abs. The release of Hb, indicating RBC lysis, was measured by enzyme-linked immunosorbent assay (ELISA). ADCC was calculated with subtraction of “no Ab” control background. All the mAbs were tested alongside RhD-pIgG, but each group of mAbs is presented in a separate figure to allow for a clear comparison. Data are the average of 2 to 4 replicates, and error bars denote SEM. Statistical analyses were conducted using 1-way ANOVA, followed by Dunnett multiple comparison test (GraphPad Prism v9). ∗ P < .05; ∗∗ P < .01.

Article Snippet: A 1:100 dilution of the supernatant was used to detect the free Hb content in each well using the Human Hb Enzyme-linked immunosorbent assay (ELISA) Kit (Elabscience catalog no. E-EL-H0415) according to the manufacturer's instructions.

Techniques: Modification, Red Blood Cell Lysis, Enzyme-linked Immunosorbent Assay, Control, Comparison