• Quantified phl p1 was used
• The sample was mixed with sample buffer and heated (5min at 95°C).
• Four different amounts of phl p1 with a C-terminal his-tag were loaded (100ng, 50ng, 20ng and 10ng) onto two 12% SDS-gel
• The electrophoresis was performed in SDS running buffer (35mA, 30min).

Immunoblot:

• Nitrocellulose membrane was used.
• The membranes were equilibrated in blotting buffer for 10 minutes.
• A semi-dry blotting device was used (125 mA, 30min).
• Blocking in 5% (w/v) skim milk in PBS-T for 30min with gentle shaking.
• Wash (3x) with PBS-T.
• The nitrocellulose membranes were cut into two pieces each and each was incubated separately:
1. Incubation with the rabbit anti-his antibody (1:10 000 in PBS-T)
2. Incubation with a mouse anti-his antibody (1:2000 in PBS-T)
3. Incubation with the same anti-his antibody (1:5000 in PBS-T)
4. Incubated with the same anti-his antibody (1:10 000 in PBS-T)
• Incubation for 45min with gentle shaking.
• Wash (3x) with PBS-T.
• Secondary antibody labelled with alkaline phosphatase (Goat-anti mouse-Fc or Goat-anti-rabbit-Fc; both 1:10 000).
• Incubation for 45min with gentle shaking.
• Wash (3x) with PBS-T.
• Equilibration of membranes with AP buffer for 10min.
• AP buffer with NBT/BCIP was added and the blots were developed in the dark for a few minutes.

Results:

P: Prestained protein marker (Pageruler, Fermentas)
1: 100ng of phl p 1
2: 50ng of phl p1
3: 20ng of phl p 1
4: 10ng of phl p 1

Hier finden Sie die beiden getesteten Antikörper:

Mouse His6-Tag Antikörper
Rabbit His6-Tag Antikörper

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