ERK 1/2 Antibody (C-9): sc-514302

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:5000) -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 (1:5000) -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

Briefly, cell lysates prepared using ice-cold radioimmunoprecipitation assay (RIPA) buffer (Millipore) was applied to IP or IB assays, with the appropriate antibodies. For the ubiquitinayltion assay in cells, 10 mM N-ethylmaleimide (NEM; Sigma-Aldrich) was subsequently added to the RIPA buffer. The cell lysates were subjected to IP with the indicated antibodies. The ubiquitin-conjugated proteins were detected by IB. For the ubiquitination assays under denaturing conditions, denatured cell extracts were prepared by resuspending cell pellets in 1 ml of denaturing buffer [50 mM tris (pH 7.5), 150 mM NaCl, 1% SDS, and 10 mM NEM] and boiled for 10 min. IPs were performed with an antibody recognizing Myc after addition of 9 ml of tris-buffered saline (TBS) buffer [50 mM tris (pH 7.5) and 150 mM NaCl] together with 0.5% NP-40 and 10 mM NEM. To detect the ubiquitinated RAS proteins, we captured the ubiquitinated proteins using an UbiQapture-Q Kit50 (Enzo Life Sciences, Switzerland) according to the manufacturer’s instructions. Cells were collected and lysed with RIPA buffer. We then added 50 μl of UbiQapture-Q matrix to the cell lysate. The sample was resuspended gently by inversion at 4 °C overnight to allow the ubiquitinated protein conjugates to bind to the affinity matrix. After centrifugation at 5000 × g for 30 s, the matrix was washed twice in phosphate-buffered saline (PBS). Extracted ubiquitinated proteins were then subjected to immunoblotting using anti-RAS (Millipore, 05-516, 1:3000) antibody. For the GST pull-down assay, bacterially expressed GST-H, K, or NRAS were purified using glutathione agarose beads (BD Biosciences). The cell lysates were incubated with the purified soluble GST fusion proteins and the pull-down samples were subjected to IB analyseis. Following primary antibodies were used for IB; anti-Ras (Millipore, 05-516, 1:3000), anti-HRas (Santa Cruz Biotechnology, sc-520, 1:500), anti-KRas (Santa Cruz Biotechnology, sc-30, 1:500), anti-NRas (Santa Cruz Biotechnology, sc-31, 1:500), anti-p-ERK (Cell Signaling Technology, #9101S, 1:1000), anti-ERK (Santa Cruz Biotechnology, sc-514302, 1:5000), anti-PCNA (Santa Cruz Biotechnology, sc-56, 1:3000), anti-N-cadherin (BD Bioscience, #610920, 1:3000), anti-αSMA (Abcam, ab7817, 1:3000), anti-Myc (Cell Signaling Technology, #2276S, 1:3000), anti-FLAG (Sigma-Aldrich, F7425, 1:3000), anti-HA (Santa Cruz Biotechnology, sc-7392, 1:3000), anti-V5 (MBL International., M167-3, 1:3000), anti-GFP (Santa Cruz Biotechnology, sc-8334, 1:3000), anti-GST (Santa Cruz Biotechnology, sc-374171, 1:3000) and anti-β-actin (Santa Cruz Biotechnology, sc-47778, 1:3000). WDR76 polyclonal antibody was generated from immunization of rabbits with partially purified WDR76 proteins (GST-WDR76 1-300; Abfrontier, Korea). The antibody was purified using ProteinA-Sepharose and a standard procedure. Secondary antibodies, horseradish peroxidase-conjugated anti-mouse antibody (Cell Signaling Technology, #7076, 1:5000) or anti-rabbit antibody (Bio-Rad, #1706515, 1:5000) were used in this study. The band signals were acquired with a LAS-4000 LCD camera coupled to MultiGauge software (Fuji).

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