Cdc42 Antibody (B-8): sc-8401

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:500) -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 (1:500) -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

ZR-75, MDA-MB-231, and MDA-MB-435s breast cells were washed with ice-cold PBS and then lysed in lysis buffer containing 10 mM Tris (pH 7.5), 150 mM NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA), 1% sodium dodecyl sulfate (SDS), 1 mM sodium orthovanadate, and a mixture of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/mL pepstatin A, and 2 μg/mL aprotinin). The lysates were sonicated for 10 s, centrifuged for 20 min at 20,000 × g, and then stored at −70°C. Equal amounts (25 μg) of the cell lysates were resolved by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes. After blocking, blots were incubated mouse anti-DDEFL1 monoclonal antibody (sc-135740; 1:500; Santa Cruz Biotechnology) or b-actin (1:1000; Zhongshan Golden Bridge Biotechnology) overnight at 4°C followed by each corresponding second antibody at room temperature for 1 h at 37°C. The results developed by ECL (Pierce Biotechnology, USA). The protein bands were then analyzed using the BioImaging System (UVP, USA). The grayscale values of DDEFL1 were normalized to the values of the corresponding b-actin band to determine the expression level of the protein. The experiments were repeated at least three times independently.

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Manufacturer protocol

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