I ran Western Blot to detect Egr-1 protein from MC3T3 cell lines, but my blot has too much unspecific bands so I can't detect protein I want. Here is my work flow, the reagents I used, and my results. Please tell me what should I do to improve my western blot results. Thanks!

  • Add 200 μl of Trypsin/EDTA per well (I cultured the cells in a 6-well plate) and incubate the plate at 37oC for 5 mins for cell detachment.
  • Add 500 μl of culture media (α-MEM) to inhibit Trypsin, collect the cell in a 1.5 ml eppendorf tube.
  • Centrifuge at 12000 rpm/3 mins/Room temperature. Aspirate the media.
  • Lyse the cell with 200 μl RIPA buffer + 2 μl Protease Inhibitor + 2 μl Phosphorylation Inhibitor. Mix by pipette and keep the mixture on ice for 20 minutes.
  • Centrifuge at 12000 rpm/15 mins/4oC. Collect ~ 180 μl of supernatant and measure the protein concentration.
  • Add loading dye and boil the protein for 20 minutes. Store the samples at -20oC.
  • Make the polyacrylamide gel 10% based on the instruction in Molecular Cloning (Sambrook). Boil the samples for 20 minutes prior loading. Ran PAGE-SDS at 150V, 30 mA, 90 minutes.
  • Transfer the protein from the gel to the nitrocellulose membrane by wet transfer techniques. Transfer at 100V, 400mA, 90 minutes, 4oC (I used ice to cool the transfer tank)
  • Block the membrane by 5% skim milk for 2 hours at RT. Wash the membrane 3 times, each time 5 minutes with TBST after blocking.
  • Incubate the membrane with primary antibody overnight at 4 degree(I used EGR1 15F7 rabbit monoclonal antibody of Cell Signalling, and β-Actin sc-47778 mouse monoclonal antibody of Santa Cruz as control). I dilute the primary antibody at 1:1000 ratio in TBST (5 μl in 5ml).
  • Wash the membrane 3 times, each time 5 minutes with TBST prior secondary antibody incubating. I used Anti-Rabbit IgG HRP Conjugate W4011 and Anti-Mouse IgG HRP Conjugate W4021 of Promega. I dilute the secondary antibody at 1:2500 ratio in TBST (2 μl in 5ml). I incubate the membrane with secondary antibody for 90 minutes at RT.
  • Wash the membrane 3 times, each time 5 minutes with TBST then I start protein detection. I used Immobilon Western Chemiluminescent HRP Substrate to detection. For β-actin, I exposed the membrane for 10 seconds. For Egr-1, I exposed the membrane up to 1 minutes. The expected sized of β-actin is 43kDA and of Egr-1 is 75kDA.
  • I can get the β-actin bands (very clear) but I can't get the Egr-1 bands.

    I add the membrane in the attachments. The dots in the Egr-1 file is the 70kDa marker.

    Thank you for spending your precious time helping me! I really appreciate that.

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