图1 Low-picogram to high-femtogram detectionTurbo GFP-His-HA-Flag was diluted in electrophoresis reducing sample buffer. Lane 1 contained 10 pg of the purified protein with serial dilutions prepared 1:1 and applied at 10 µL/well. After electrophoresis, proteins were transferred to nitrocellulose membrane (Cat. No. 88018) using the Pierce™ Power Blotter (Cat. No. 22834) and Pierce 1-Step Transfer Buffer (Part No. 84731). The membrane was blocked with SuperBlock in TBST Buffer (Part No. 37536). Then the membrane was incubated with Anti-His (Part No. MA1-21315) at 1 µg/mL, followed by incubation with Goat anti-Mouse Horseradish Peroxidase Conjugate (Part No. 32430) at 100 ng/mL. SuperSignal West Pico PLUS substrate (Part No. 34577) was used for detection. A thirty second exposure was acquired on the Thermo Scientific MYECL Imager (Part No. 62236) and the image was inverted and contrasted (black = 55,000, white = 65,535, gamma = 1.0).

HDAC1 and Rab9 detection in HeLa lysates (lane 1: 20 µg total protein; lanes 2-6: serially diluted 1:1) was performed using SuperSignal West Pico PLUS chemiluminescent substrate. The blots were developed using either iBind Flex (top row) or traditional Western blotting (bottom row) protocols. The HDAC1 blots were developed using an anti-HDAC1 polyclonal antibody (Cat. No. PA1-860) followed by HRP-conjugated goat anti-rabbit IgG secondary antibody (Cat. No. 31460), and the Rab9 blots were developed using an anti-Rab9 monoclonal antibody (Cat. No. MA3-067) followed by HRP-conjugated goat anti-mouse IgG secondary antibody (Cat. No. 31430). For the traditional western blotting protocol, the primary antibody was incubated for 1 hour at room temperature and the secondary antibody was incubated for 30 minutes at room temperature. Images were captured using the myECL Imager.

图3 Product comparisonDetection of the indicated targets was performed using 2-fold serial dilutions of HEK293 or HeLa cell lysates, starting with the amount indicated in parentheses above. Following separation by SDS-PAGE, proteins were transferred to either PVDF (Cat. No. 88518) or nitrocellulose (Cat. No. 88018) membranes using the Pierce™ Power Blotter (Cat. No. 22834) and 1-Step Transfer Buffer (Cat. No. 84731). The membranes were blocked with 5% non-fat dry milk dissolved in Pierce 20X TBS Tween™ 20 Buffer (Cat. No. 28360), and incubated with antibodies against beta-Catenin (Cat. No. MA1-300), eIF4E (Cat. No. MA1-089), RSK2 (Cat. No. MA5-15920), or Ezrin (Cat. No. MA5-13862), followed by incubation with Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP Conjugate (Cat. No. 31430) at a concentration of 20 ng/mL. Chemiluminescent detection and substrate comparison was performed following a 5-minute incubation with either SuperSignal West Pico PLUS™ or Bio-Rad Clarity™ substrates. Signal was captured using film.