The dot blot was performed as described previously.40 (link) 2 ul of dissolved needles and soluble control were pipetted and dried onto a nitrocellulose membrane for 15 min. The membrane was blocked overnight with 5% powdered milk (Sigma-Aldrich) in TBS–0.1% Tween 20 (TBST) at 4°C overnight. The proteins were detected using 6x-His epitope tag primary antibodies raised in mice (ThermoFisher). After 2 hrs, the membrane was washed three times with TBST and incubated with goat anti-mouse secondary antibodies conjugated to alkaline phosphatase at 1:5000 dilution. The proteins were then detected using Western Blue Stabilized Substrate for alkaline phosphatase (Promega cat # S3841) and imaged after 5 min incubation.
Protocol detail
Quantitative Dot Blot Analysis of Proteins
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Alkaline phosphatase
Anti antibodies
Antibodies
Dilution
Dot blot
Epitope
Goat
Membrane
Milk
Mouse
Needles
Nitrocellulose
Promega
Proteins
Tween 20
Corresponding Organization : VA Maryland Health Care System
Other organizations : National Institute of Allergy and Infectious Diseases
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Variable analysis
independent variables
- Dissolved needles
- Soluble control
- Proteins detected using 6x-His epitope tag primary antibodies
- Nitrocellulose membrane
- Blocking with 5% powdered milk in TBST
- Incubation time with primary antibodies (2 hrs)
- Washing with TBST
- Incubation with secondary antibodies conjugated to alkaline phosphatase (1:5000 dilution)
- Substrate used for alkaline phosphatase detection (Western Blue Stabilized Substrate)
- Incubation time for substrate (5 min)
- Soluble control
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