Protocols:

 

Protein/Phosphoprotein Detection in Immunoblots

 

1. Protein Transfer to a Membrane
  • For protein/phosphoprotein detection we recommend to use PVDF membranes, e.g. PALL BioTraceTM PVDF (#BTPV 500-07).
  • Transfer proteins to a PVDF membrane by the following Western Blotting procedure
Transfer-Buffers
Anode Buffer 1 (AP1): 300 mM Tris, pH 9.5
Anode-Buffer 2 (AP2):  30 mM Tris, pH 9.5
Cathode-Buffer (CB):    40 mM 6-Aminohexanoic acid,
                                  20% (v/v) Methanol

  • The blotting sandwich contains the following layers :
Anode (+)
             1x filter paper moisted with AB1
              1x filter paper moisted with AB2
      PVDF membrane
      Gel
            2x filter paper moisted with CB
Cathode (-)
  • PVDF membrane, e.g PALL BioTraceTM PVDF (#BTPV 500-07)
  • Filter paper, e.g. Whatman Schleicher&Schuell gel Blotting Paper GB002
IMPORTANT: Make sure to remove all air bubbles between the gel and the PVDF membrane and the filter layers.
  • Blotting time per gel at 5.5 mA/cm2 15 min ( 10% mini gel 0.75 mm thick, protein size 30-200 kDa)
  • After blotting wash the PVDF membrane for at least 30 min at 15-22C with the General Washing Buffer, e.g. 2x PBS/0.1% (v/v) Tween 20.
2. Blocking of Nonspecific Binding Sites
  • Block blots for at least 2 hours with Blocking Solution. Blocking overnight is also possible (add phosphatase-inhibitors, e.g. sodiumfluoride, sodiumvanadate if the assay targets are phosphorylated epitopes).
  • Wash three times 5 min with 2x PBS/0.1% (v/v) Tween 20.
General Blocking Solution
We recommend a casein/Tween 20 based blocking solution (e.g. nanoTools product #3031-500/CPPT or #3031-3000/CPPT) for the use with phosphorylation-site specific antibodies and antibodies that are not directed to phosphorylated epitopes, unless otherwise stated in the datasheet.

Alternative Blocking Solution
This blocking solution is recommended for the use with phosphorylation-site specific antibodies that cannot be used with a casein based blocking solution. These are especially the phosphoserine/threonine-specific antibodies that are included in our Phosphoserine and Phosphothreonine Detection Kits (see list below).
Phosphoserine-specific antibodies: PSER-1C8; -4A3; -4A9; -4H4; -7F12; -16B4
Phosphothreonine-specific antibodies: PTHR-1E11; -4D11; -14B3
For these antibodies, it is essential to avoid the use of blocking reagents containing phosphoproteins (e.g. milk or casein), since this will lead to competition of the highly phosphorylated casein with the phosphoserine-/ phosphothreonine-specific monoclonal antibodies. Application of casein or milk may lead to significant decrease or loss of signal intensity due to reaction of the primary antibody with the blocking/diluting agent. We therefore recommend to replace casein by 2% (w/v) BSA.

3. Antibody incubation, Washing and Detection

General Washing Buffer
2x PBS or 2x TBS with 0.1% (v/v) Tween 20

Incubation temperature: We recommend to perform all steps at temperatures between 15 and 22C. Incubation at lower temperatures requires prolonged incubation time; higher temperatures will significantly increase background staining.

Expermiental precedure:
  • Incubate for 1 hour at 15-22C with primary antibody diluted in Blocking Solution.
  • Wash three times 5 min with General Washing Buffer.
  • Incubate for 45 min at 15-22C with secondary antibody conjugate (HRPO) diluted in Blocking Solution.
  • Wash five times 5 min with General Washing Buffer.
  • Develop by ECL (Note: The blots should be dried carefully with filter paper to remove all remaining ECL liquid in order to reduce background staining).




Mouse