Antibody validation and detection of pro-BDNF in human skeletal muscle. Validation of the Novus (NB100-98682) antibody in its ability to detect pro-BDNF in different samples. (A) The gel is loaded according to the following: (1) human skeletal muscle homogenate 22 µg protein; (2) human platelet extract 20 µg protein; (3) human platelet poor plasma 20 µg protein; (4) human type I fiber pool 4 µg protein; (5) human type II fiber pool 4 µg protein; (6) recombinant mBDNF 0.25 ng; and (7) recombinant pro-BDNF 0.25 ng. The antibody is able to detect pro-BDNF at 28 kDa. The antibody also detects mBDNF at 14 kDa, found in the platelet extract that is known to be highly enriched with mBDNF. (B) In this gel, blotted with NB100-98682: is (1) human plasma samples with 25 µg of protein loaded; (2) human platelet extract 20 µg protein; (3) human skeletal muscle homogenate 20 µg protein; (4) mouse Soleus muscle homogenate 20 µgprotein; (5) mouse EDL muscle homogenate 20 µg protein; (6) HeLa cell lysate (Santa Cruz Biotechnology sc-2200) 35 µg protein, serving as a negative control lysate; and (7) mouse IgG 0.1 µg, serving as a negative control. (C) Schematic overview of BDNF metabolism. (D) Skeletal muscle levels of pro-BDNF (pg mg⁻¹ dry muscle) for all the 117 muscle samples analyzed in the study. (E) Validation of the Abcam (ab108319) antibody to detect mBDNF in different samples: (1) human skeletal muscle homogenate 40 µg protein; (2) human skeletal muscle homogenate 20 µg protein; (3) human type I fiber pool 25 µg protein; (4) human type II fiber pool 25 µg protein; (5) human plasma protein 15 µg of protein; (6) human plasma protein 30 µg of protein; (7) human platelet extract 4 µg protein; (8) human platelet extract 8 µg protein; (9) human platelet extract 12 µg protein; (10) HeLa cell lysate (Santa Cruz Biotechnology sc-2200) 35 µg protein, serving as a negative control lysate; (11) mouse IgG 0.1 µg, serving as a negative control; and (12) recombinant mBDNF 0.25 ng.