agarose bound con a resin  (Vector Laboratories)

Journal: Journal of Extracellular Vesicles

Article Title: Proteomic Analysis of Extracellular Vesicles Identifies CDCP1 as Critical Metastasis‐Related Glycoprotein in Lung Cancer

doi: 10.1002/jev2.70128

Figure Lengend Snippet: EVs glycoprotein enrichment and proteomic analysis. (A) Workflow for proteome identification of EV total proteins and glycoproteins. (B) Principal component analysis of 95C and 95D EVs total proteins (T‐p) and glycoproteins (G‐p). (C) Venn diagram of EVs total proteins and glycoproteins. (D) Pearson correlation coefficient matrix plot of multiple groups. The correlation coefficient was represented by a color scheme from white (negative correlation) to blue (positive correlation). (E) The volcano plot represents the quantitative analysis of N‐glycoproteins in 95D EVs versus 95C EVs. Orange represents proteins that were at least 2‐fold upregulated ( p < 0.05); blue represents proteins that were at least 2‐fold downregulated ( p < 0.05). (F) Distribution plot of the number of N‐glycosylation sites of 95C and 95D EVs. (G‐I) Comparison of the ranking, relative quantity, and unique peptides of three typical glycoproteins before and after ConA enrichment. GO analysis (J) and KEGG pathway enrichment (K) of differentially expressed glycoproteins in EVs. BP, biological process; CC, cellular component; MF, molecular function.

Article Snippet: Briefly, for 1 mg EVs protein, 1 mL of agarose bound lectin ConA (Vector Laboratories, AL‐1003) was packed into a 2 mL disposable screw end‐cap spin column with filter (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Glycoproteomics, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Identification of two rate-limiting steps in the degradation of partially folded immunoglobulin light chains

doi: 10.3389/fcell.2022.924848

Figure Lengend Snippet: Both domains are reduced in the retrotranslocated NS1 protein. (A) Cells were transfected with either the cytosolically expressed ΔssNS1 or NS1. After 24 h, cells were treated with (+) or without (-) MG132, and cell lysates were prepared in NP-40 buffer containing NEM. Samples were analyzed by non-reducing SDS-PAGE-coupled western blotting with goat anti-mouse κ antisera. Migration of the various oxidation species (ox0, ox1, ox2) is indicated. (B) Cells expressing NS1-N100 were treated with (+) or without (-) MG132 for 3.5 h prior to lysis. Lysates were prepared and analyzed under reducing or non-reducing conditions as indicated. A portion of the lysate from cells not treated with MG132 was digested with Endo H (+) to determine the migration of the deglycosylated ox1 and ox2 species. An unidentified species (??) generated by proteasomal inhibition observed on non-reducing gels that co-migrated with reduced, deglycosylated NS1-N100 is indicated with a red dotted line. (C) 293T cells expressing NS1-N100 were incubated with MG132 (+) or DMSO (-). Cells were lysed in NP-40 lysing buffer supplemented with NEM, and a portion of each was kept as an input. The remaining lysate was absorbed with Con A-conjugated beads. Equivalent fractions of the Con A-unbound and the sample was eluted from the Con A beads (bound) were analyzed by SDS-PAGE conducted under non-reducing conditions and blotted with anti-κ antisera. Bands corresponding to the various redox states are indicated as is their glycosylation status. A red dotted line indicates the species generated by proteasomal inhibition observed panel B is in fact fully reduced and non-glycosylated.

Article Snippet: Concanavalin A (Con A) beads (AL-1003, VECTOR LABORATORIES, Burlingame, CA) were used to separate glycosylated and non-glycosylated species.

Techniques: Transfection, SDS Page, Western Blot, Migration, Expressing, Lysis, Generated, Inhibition, Incubation