If you are looking for Western blot antibody against a specific gene, type in the gene name in the box above and hit the search button. In the resultant gene webpage, select 'antibody' link for the correct gene to go to the antibody webpage, and use the 'Western blot' filter as application.

Application: Immunoblotting can be used to determine the presence of specific protein, and its size/molecular weight (if the lysates or extracts are separated under denaturing condition). It can also be used to determine the relative abundance of a specific protein, although the absolute quantity can not be determined. In addition, if phospho antibody is used as the primary antibody, the phosphorylation status of a specific protein can be determined, usually after the protein has been immunoprecipitated.

1. Cell lysates or other source of protein to be tested
2. Immunoblotting membrane, nitrocellulose or PVDF
3. Primary antibody
4. Secondary antibody-enzyme conjugate
5. Detection system (eg. ECL)
6. Buffers
   1. Lysis buffer:
      • 0.15 M NaCl
      • 5 mM EDTA, pH 8
      • 1% Triton X100
      • 10 mM Tris-Cl, pH 7.4
      • Just before using add: 1:1000 5 M DTT, 1:1000 100 mM PMSF in isopropanol, 1:1000 5 M e-aminocaproic acid
   2. 2x sample buffer:
      • 130 mM Tris-Cl, pH8.0
      • 20% (v/v) Glycerol
      • 4.6% (w/v) SDS
      • 0.02% Bromophenol blue
      • 2% DTT
   3. 10x Running buffer:
      • Trizma base (0.25 M)
      • Glycine (1.92 M)
      • SDS (1%)--add last, Do not adjust the pH
   4. 10x Blotting buffer:
      • Trizma base (0.25 M)
      • Glycine (1.92 M)
      • pH should be 8.3; do not adjust
      • To make 2 L of 1x Blotting buffer:
        • 400 ml Methanol
        • 200 ml 10x Blotting buffer
        • 1400 ml water
   5. Blocking buffer:
      • 3% Bovine serum albumin (Fraction V)
      • Make up in PBS and sterile filter. Then add 0.05% Tween 20. Keep at 4°C to prevent bacterial contamination.
   6. Stripping buffer: (sterile filter solution and keep at 4°C)
      • 0.2 M Glycine, pH 2.5
      • 0.05% Tween 20

1. Preparation of cell lysates
   1. Collect cells by trypsinization and spin.
   2. Lyse the pellet with 100 µl lysis buffer on ice for 10 min. For 500,000 cells, lyse with 20 µl.
   3. Spin at 14,000 rpm (16,000 g) in an Eppendorf microfuge for 10 min at 4°C.
   4. Transfer the supernatant to a new tube and discard the pellet.
   5. Determine the protein concentration (Bradford assay, A280, or BCA)
   6. Take x µl (= y µg protein) and mix with x µl of 2x sample buffer.
   7. Boil for 5 min.
   8. Cool at RT for 5 min.
   9. Flash spin to bring down condensation prior to loading gel.
2. Running the gel
   1. After flash spinning the samples, load into the wells.
   2. Be sure to use markers.
   3. Run with constant current (35 - 37 mA with voltage set at > 300 V).
   4. Usual running time is about 2.5 hr.
3. Membrane transfer
   1. Assemble "sandwich" for Bio-Rad's Transblot.
   2. Prewet the sponges, filter papers (slightly bigger than gel) in 1x Blotting buffer. Sponge - filter paper - gel - membrane - filter paper - sponge
   3. Transfer for 1 hr at 1 amp at 4°C on a stir plate.      Bigger proteins might take longer to transfer.      For the Mini-Transblot, it's 100 V for 1 hr with the cold pack and prechilled buffer.
   4. When finished, immerse membrane in Blocking buffer and block overnight.
4. Detection
   1. Incubate with primary antibody diluted in Blocking buffer for 60 min at room temp.
   2. Wash 3 x 10 min with 0.05% Tween 20 in PBS.
   3. Incubate with secondary antibody diluted in Blocking buffer for 45 min at room temp.
   4. Wash 3 x 10 min with 0.05% Tween 20 in PBS.
   5. Expose the membrane to an appropriate substrate for visulization of the blot.

mouse antibody. Labome product database includes 2682 mouse genes with Western blot monoclonal antibodies, and 7533 mouse genes have Western blot polyclonal antibodies.

rat antibody. Labome product database includes 1908 rat genes with Western blot monoclonal antibodies, and 6287 rat genes have Western blot polyclonal antibodies.

rabbit antibody. Labome product database includes 1606 genes with rabbit-derived Western blot monoclonal antibodies, and 19973 genes with rabbit-derived Western blot polyclonal antibodies.

mouse antibody. Labome product database includes 10553 genes with mouse-derived Western blot monoclonal antibodies, and 7446 mouse-derived Western blot polyclonal antibodies.

rat antibody. Labome product database includes 1282 genes with rat-derived Western blot monoclonal antibodies.

goat antibody. Labome product database includes 18429 genes with goat-derived Western blot polyclonal antibodies.

chicken antibody. Labome product database includes 1525 genes with chicken-derived Western blot polyclonal antibodies.