Vimentin Antibody (5G3F10): sc-66002

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:1500) --Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Protocol tips
-Mouse (1:1500) --Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink can fluoresce and cause background.

Publication protocol

After 48 h of SW1990 and PANC-1 cell transfection, the cells (1 × 106 cells) were washed with PBS two times and then lysed with 150 μL of RIPA lysis buffer with a 1% phenylmethanesulfonyl fluoride (PMSF) mixture for 30 min. Thereafter, the lysates were centrifuged at 4 °C at 12000 g for 15 min, and the supernatant was used. An enhanced BCA protein assay kit was used to detect the protein concentration (Beyotime, Shanghai, China). A 25 μg standard protein solution was diluted to 20 μL with PBS, and 5 μL of protein buffer was added to an OSE-DB-01 metal bath (TIANGEN Biotech, Beijing, China) for 10 min at 95 °C. The obtained protein samples were then loaded into a 10% SDS-PAGE separation gel to separate the total proteins, followed by transfer to a polyvinylidene fluoride (PVDF) membrane. Then, the membrane was sealed with 5% skimmed milk at room temperature for 1 h, followed by incubation overnight at 4 °C with the primary antibody at a concentration of 1:3000 for SMAD4 (EP618Y, Abcam, U.S.A.), 1:1500 for Vimentin (5G3F10, Santa Cruz, USA), 1:2000 for E-cadherin (G-10, Santa Cruz, USA) or 1:1000 for GAPDH (AB9483, Abcam, U.S.A.). The appropriate secondary antibody coupled to horseradish peroxidase (HRP) was then applied to the membrane at a concentration of 1:4000, followed by incubation at room temperature for 1 h. The protein bands were detected by enhanced chemiluminescence (Thermo Fisher Scientific, USA).

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