User Test for HIS-Tag Antibodies

Scientists from the Fraunhofer Institute (IME) have tested a rabbit HIS6-tag antibody and a mouse HIS6-tag antibody. Take a look at the results here.

SDS-Page:

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:

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

Take a look at the two tested antibodies:

Mouse His6-Tag Antibody
Rabbit His6-Tag Antibody

Antibodies for the Research Area "Tags and Labels": Show antibodies

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23.01.2007 | Tim Hiddemann

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