Sigmafast dab with metal enhancer tablets

In order to confirm the specificity of VHHs for CB, Western blot analysis was performed. Ten micrograms of CB were reduced, electrophoresed on 12.5% SDS-PAGE, and transferred to a nitrocellulose membrane. Reactive sites were blocked with 5% skimmed milk in TBS buffer (TBSM) at 4 °C overnight. After being washed three times with TBS/0.1% Tween 20 (TBST), the strips were incubated with 0.2 mg/mL of each anti-crotoxin VHH (KF498602, KF498603, KF498604 and KF498605) overnight. The strips were washed again with TBST and incubated with rabbit anti-llama IgG2/IgG3 (1:1000 in 5% TBSM) overnight. After being washed, the samples were incubated with peroxidase conjugated mouse anti-rabbit IgG (1:3000 in 5% TBSM) for 6 h. The strips were washed, and the reactive signals were detected after incubation with hydrogen peroxide in diaminobenzidine (DAB) solution (SIGMAFAST™ DAB with Metal Enhancer Tablets, Sigma-Aldrich). Llama immune serum (1:1000) was used as the positive control and an anti-BthTX VHH (KC329718-0.2 mg/mL) [42 (link)], as the negative control.

For the Western blot (WB) analysis, 10 μg of BthTX-I and BthTX-II were reduced, electrophoresed on a 15% SDS-PAGE, and transferred to a nitrocellulose membrane (Amersham Life Science). Reactive sites were blocked with 5% skimmed milk in TBS buffer (TBSM) at 4°C overnight. After washing with TBS/0.1% Tween 20 (TBST), the strips were incubated with 0.2 mg/mL of each anti-BthTX VHH (KF498607, KF498608, KC329715 and KC329718) overnight. Strips were washed with TBST and incubated with anti-His antibody (GE Healthcare) (1:3000 in 5% TBSM) for 16 h. After washing, the samples were incubated with HRP-conjugated anti-mouse IgG produced in goat (1:3000 in 5% TBSM). Strips were washed, and the reactive signals detected after incubation with hydrogen peroxide in diaminobenzidine (DAB) solution (SIGMAFAST™ DAB with Metal Enhancer Tablets, Sigma-Aldrich). Llama postimmunization serum (1:1000) was used as positive control. The negative control was performed with the llama pre-immune serum (1:1000).