Haynes:WestBlot: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Line 16: | Line 16: | ||
* 10x PBS | * 10x PBS | ||
* Tween-20 (e.g., G-Biosciences 786-517) | * Tween-20 (e.g., G-Biosciences 786-517) | ||
'''EQUIPMENT''' | '''EQUIPMENT''' | ||
* Electroblotting chamber (e.g. Invitorgen XCell SureLock) | * Electroblotting chamber (e.g. Invitorgen XCell SureLock) | ||
* Blotting sponge pads (e.g. Invitrogen EI9052) - usually included with electroblotting unit | |||
Line 33: | Line 33: | ||
''Instructions for a wet chamber'' | ''Instructions for a wet chamber'' | ||
# Remove the gel from the mold, trim the top and bottom, and soak in Western blot transfer buffer “back” side up. | # Remove the gel from the mold, trim the top and bottom, and soak in Western blot transfer buffer “back” side up. | ||
# In a small flat container of '''1x Transfer Buffer''', wet two | # In a small flat tupperware-like or glass container of '''1x Transfer Buffer''', wet two '''blotting sponge pads'''. | ||
# Wet one piece of '''blotting paper''' in the '''1x Transfer Buffer'''. Place it on top of a wet sponge pad. | |||
# | |||
Revision as of 15:21, 24 March 2015
Western Blot
by Karmella Haynes, 2015
Principle: Proteins that have been separated by size on a polyacrylamide gel (by PAGE)are transferred onto a solid paper-like membrane, such as nitrocellulose. One specific protein is visualized by incuabating the membrane with labeled antibodies, then imaging the membrane. A visible band will appear that corresponds to the size of the protein.
MATERIALS
- PAGE gel containing electrophoresed protein samples
- Tris-glycine concentrate [250 mM Tris; 1.92 M glycine; pH approx. 8.3] (see recipe)
- Methanol
- Blotting paper/ "filter" paper (e.g. Bio-Rad Nitrocellulose & filter sets 1620212)
- Nitrocellulose or PVDF membrane (e.g., Bio-Rad Nitrocellulose & filter sets 1620212)
- 10x PBS
- Tween-20 (e.g., G-Biosciences 786-517)
EQUIPMENT
- Electroblotting chamber (e.g. Invitorgen XCell SureLock)
- Blotting sponge pads (e.g. Invitrogen EI9052) - usually included with electroblotting unit
PROCEDURE
Prepare 1x Transfer Buffer [12.5 mM Tris; 96 mM glycine; 10% methanol]
- Add ~200 mL dH2O, 25 mL Tris-glycine concentrate, and 50 mL methanol to a 500 mL bottle. Swirl to mix.
- Fill up to 500 mL with dH2O (+ ~225 mL dH2O). Note: deionized water from the tap is sufficient.
Prepare a gel and filter stack
Instructions for a wet chamber
- Remove the gel from the mold, trim the top and bottom, and soak in Western blot transfer buffer “back” side up.
- In a small flat tupperware-like or glass container of 1x Transfer Buffer, wet two blotting sponge pads.
- Wet one piece of blotting paper in the 1x Transfer Buffer. Place it on top of a wet sponge pad.
Prepare the electrophoresis chamber
Prepare 1x PBS-tween [? mM Na2HPO4; ? mM NaH2PO4; ? nmM NaCl; 0.1% Tween-20; pH ~7.2]
- Add ~200 mL dH2O, 50 mL 10x PBS, and 0.5 mL Tween-20 to a 500 mL bottle. Swirl to mix.
- Fill up to 500 mL with dH2O (+ ~249.5 mL dH2O). Note: deionized water from the tap is sufficient.
HELPFUL RESOURCES
- Electroblotting wet transfer: Video - How to perform a traditional wet protein transfer using the XCell SureLock® Blot Module