Bcl-2 Antibody (100): sc-509

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:1000) -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 (1:1000) -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

Attached cells were washed twice with ice-cold PBS and then lysed with lysis buffer for 30 min on ice. Total protein content (for cell and tissue samples) was determined using a BCA protein assay kit (ab102536, Abcam, Cambridge, UK). Equal amounts of proteins were resolved by 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk in TBS-T for 1 h and incubated with specific primary antibodies (1:1000) at 4 °C with gentle shaking overnight. Membranes were washed three times with TBS-T, reacted with secondary antibodies conjugated to HRP (1:2000; ab205719 and ab205718, Abcam, Cambridge, UK), and antibody:protein complexes detected by enhanced chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.). Data obtained from three independent experiments were analyzed with ImageJ software (v 1.52p).

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