Western blotting was performed as described previously [36 (link)]. In brief, proteins were denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system (Invitrogen, Carlsbad, CA). After transferring, the Immuno-blot PVDF Membrane (Bio-Rad, Hercules, CA) was blocked for 1 h and then probed using anti-STAT3 or anti-pSer727 STAT3 (Cell signaling Technology, Inc., Beverly, MA) overnight at 4 C with constant shaking. After washing, membrane was then incubated with an anti-mouse (for STAT3 antibody, Santa Cruz Biotechnology, Santa Cruz, CA) or anti-rabbit (for pSer727 STAT3 antibody, Amersham Biosciences, Piscataway, NJ) IgG-HRP secondary antibody. All antibodies were diluted in blocking buffer. For immunodetection, membrane was incubated with enhanced chemiluminescence solutions per the manufacturer’s specifications (Amersham Biosciences, Piscataway, NJ), and exposed to Hyperfilm ECL (Denville Scientific Inc., Metuchen, NJ).
Sds page system
Manufactured by Thermo Fisher Scientific
The SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) system is a laboratory equipment used to separate proteins based on their molecular weight. It employs an electric field to move charged proteins through a polyacrylamide gel, allowing for the visualization and analysis of protein samples.
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Lab products found in correlation
3 protocols using sds page system
1
Western Blotting Quantification of STAT3 Phosphorylation
2
Gelatin Zymography for MMP-2 and MMP-9
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MMP-2 and MMP-9 enzymatic activity was determined using a 10% gelatin zymography sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) system (Invitrogen) according to the manufacturer's instruction. Briefly, conditioned medium was mixed with 2x nonreducing sample buffer and separated on a 10% polyacrylamide gel containing 0.1% gelatin. After electrophoresis, the gel was washed and incubated in Zymogram Renaturing Buffer for 30 min at room temperature with gentle agitation to renature the gelatinases. After washing twice, the gel was incubated for 18 h in Zymogram Developing Buffer, and subsequently stained by SimplyBlue™ SafeStain. Blue-stained bands were visible on a clear gel after destaining with water. An image of the gel was detected with the Gel Doc XR system (Bio-Rad Laboratories, Hercules, CA).
3
Recombinant mEpoR Protein Expression
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The cDNA encoding residues S212-P259 of the mEpoR were synthesized by Genscript. Expression vector such as pET-29b plasmid was purchased from Merck. The SDS-PAGE system was purchased from Invitrogen. Protein sample loading dye, molecular weight standards were purchased from Bio-rad. Bl21 (DE3) competent cells were purchased from StrataGene. β-D-1-thiogalactopyranoside (IPTG), Dithiothreitol (DTT) and detergents including dodecylphosphocholine (DPC), deuterated DPC (D-DPC) were purchased from Anatrace or Avanti. The 15NH4Cl, 13C-glucose and D2O were purchased from Cambridge Isotope Laboratories. All other chemicals were purchased from Sigma.
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