claudin-1 Antibody (XX7): sc-81796

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

Whole-cell lysates were denatured at 95 °C for 5 min, and proteins were then separated in a NuPAGE 4–12% Bis-Tris gel (Life Technologies) in NuPAGE Mes or Mops SDS running buffer (Life Technologies). Gels were electroblotted onto a nitrocellulose membrane (Life Technologies) in NuPAGE transfer buffer (Life Technologies). Membranes were incubated at 4 °C overnight with appropriate primary antibodies diluted in blocking buffer [2% blocking agent (GE Healthcare) in 0.05% TBS-Tween] and subsequently washed five times for 5 min with 0.05% TBS-Tween. Membranes were then incubated with HRP-labeled secondary antibody (Sigma) for 1 h at room temperature. After five washing steps with 0.05% TBS-Tween and an additional washing step in TBS, immunoreactive proteins were detected by using ECL Advance reagent (GE Healthcare). For Fig. 4 C and D and Figs. S3C, S6 A, B, and D, and ​andS7,S7, cells were harvested by using RIPA Buffer (Sigma-Aldrich) with cOmplete protease inhibitor mixture (Sigma-Aldrich). Western blots were performed by using Wes Split Buffer Master Kit (ProteinSimple) on the “Wes” automated Western blot instrument (ProteinSimple) per the manufacturer's instructions.

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Manufacturer protocol

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