Journal: Scientific Reports

Article Title: Spiders’ digestive system as a source of trypsin inhibitors: functional activity of a member of atracotoxin structural family

doi: 10.1038/s41598-023-29576-y

Figure Lengend Snippet: Purification of Nephilingis cruentata MMD inhibitors. ( A ) Bovine trypsin inhibitory assay with the eluted fractions from the ionic-exchange batch separation and respective controls. (C1): control assay of bovine trypsin with 25 mM of ammonium bicarbonate at pH 8.5; (C2): control assay of bovine trypsin 25 mM of ammonium bicarbonate containing 1 M NaCl at pH 8.5; (C3): control assay of bovine trypsin with 25 mM of ammonium bicarbonate at pH 3.5; (1): Fraction eluted with 25 mM of ammonium bicarbonate at pH 8.5; (2): Fraction eluted with 25 mM of ammonium bicarbonate with 1 M NaCl at pH 8.5; (3): Fraction eluted with 25 mM of ammonium bicarbonate at pH 3.5. Inhibitory activity was measured using bovine trypsin 12 ng/µl as enzyme source and (0.1 mM) ZFRMCA as substrate (N = 3). ( B ) Chromatographic separation profile (UV at 214 nm) of fraction 2 (inhibitory active fraction against bovine trypsin), using a C18 column. Elution was performed by a linear gradient of 5% to 90% of solution B in 35 min. ( C ) SDS-PAGE in a 4–20% polyacrylamide gel of chromatographic peaks (P1-P6). Lane S molecular mass marker (97–14 kDa); lanes respectively: 1: P1; 2: P2; 3: P3; 4: P4; 5: P5; and 6: P6. The red arrow shows a single band detected by Coomassie blue G-250R. The approximate mass for the band on line 4 is 28.78 kDa calculated by gel relative migration.

Article Snippet: The samples eluted from chromatography on RP-HPLC (10 μg of protein) were loaded on 4–20% gradient SDS- PAGE gel kit Biocompare ( www.biocompare.com ) in reducing conditions.

Techniques: Purification, Control, Activity Assay, SDS Page, Marker, Migration

Journal: BMC Physiology

Article Title: Sterol carrier protein-x gene and effects of sterol carrier protein-2 inhibitors on lipid uptake in Manduca sexta

doi: 10.1186/1472-6793-10-9

Figure Lengend Snippet: Expression of MsSCP-x and MsSCP-2 . (A) The semi-quantitative RT-PCR shows that mRNA levels from different tissues at different developmental stages of M. sexta . Lane A: Day 1-4 th instar; lane B: Day 3-4 th instar; lane C: Day 1-5 th instar; lane D: Day 4-5 th instar; lane E: Day 1-wandering stage; lane F: Day 1-pupal stage. The Manduca rpS3 gene was used as internal standard for the RT-PCR. Only 30 cycles of PCR were performed to avoid saturating the signals. (B). Proteins from different tissues of Day 3-4 th instar M. sexta larva were loaded 20 μg per lane except lane 8 that only had 10 ng purified recombinant MsSCP-2. Protein was analyzed on 4-20% gradient SDS PAGE. Lane 1: protein size marker; Lane 2: hemolymph; Lane 3: midgut; Lane 4: hindgut; Lane 5: fat body; Lane 6: muscle; Lane 7: epidermis; Lane 8: recombinant MsSCP-2.

Article Snippet: Proteins (20 μg) from each sample were resolved on a 4-20% pre-casted gradient SDS PAGE gel (ISC Bioexpress, Kaysville, UT, USA) and transferred onto a Hybond-C extra membrane (Amersham BioScience, Piscataway, NJ, USA) at 18 volts overnight in Tris-glycine transfer buffer (0.303% Tris base, 1.44% glycine, 20% methanol) as described [ ].

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Purification, Recombinant, SDS Page, Marker