Haynes:WestBlot: Difference between revisions

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PROCEDURE
PROCEDURE


'''Prepare 1x Transfer Buffer'''<br>
'''Prepare 1x Transfer Buffer''' [12.5 mM Tris; 96 mM glycine; 10% methanol]<br>
[12.5 mM Tris; 96 mM glycine; 10% methanol]
# Add ~200 mL dH<sub>2</sub>O, 25 mL Tris-glycine concentrate, and 50 mL methanol to a 500 mL bottle. Swirl to mix.
# Add ~200 mL dH<sub>2</sub>O, 25 mL Tris-glycine concentrate, and 50 mL methanol to a 500 mL bottle. Swirl to mix.
# Fill up to 500 mL with dH<sub>2</sub>O (+ ~225 mL dH<sub>2</sub>O). ''Note: deionized water from the tap is sufficient.''  
# Fill up to 500 mL with dH<sub>2</sub>O (+ ~225 mL dH<sub>2</sub>O). ''Note: deionized water from the tap is sufficient.''  


'''Prepare 1x PBS-tween'''<br>
'''Prepare 1x PBS-tween''' [? mM Na2HPO4; ? mM NaH2PO4; ? nmM NaCl; 0.1% Tween-20; pH ~7.2]<br>
[? mM Na2HPO4; ? mM NaH2PO4; ? nmM NaCl; 0.1% Tween-20; pH ~7.2]
# Add ~200 mL dH<sub>2</sub>O, 50 mL 10x PBS, and 0.5 mL Tween-20 to a 500 mL bottle. Swirl to mix.
# Add ~200 mL dH<sub>2</sub>O, 50 mL 10x PBS, and 0.5 mL Tween-20 to a 500 mL bottle. Swirl to mix.
# Fill up to 500 mL with dH<sub>2</sub>O (+ ~225 mL dH<sub>2</sub>O). ''Note: deionized water from the tap is sufficient.''  
# Fill up to 500 mL with dH<sub>2</sub>O (+ ~225 mL dH<sub>2</sub>O). ''Note: deionized water from the tap is sufficient.''  
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# 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.
#  
#  
# Add protein samples to 1.5 mL tubes. Dilute the stock proteins in an appropriate buffer. Make sure the protein sample volume = the final volume - (concentrated sample buffer volume + 1.0 μL 1M DTT)
 
## Example 1: 20 μL final loading volume - (5.0 μL 4x sample buffer + 1.0 μL 1M DTT) = 14.0 μL protein
## Example 1: 20 μL final loading volume - (10.0 μL 2x sample buffer + 1.0 μL 1M DTT) = 9.0 μL protein
# Calculate the number of lanes used = protein samples + protein standard(s). If this is less than the total number of lanes, prepare "dummy" samples (sample buffer + DTT + water) to fill empty wells
# Heat samples, excluding the protein standard(s), @ 100°C/ 5 min. Cool at room temp.


'''Prepare the electrophoresis chamber'''
'''Prepare the electrophoresis chamber'''
# Prepare the running buffer: dilute the buffer to make 500 mL of 1x running buffer.
#  
## Note: 500 mL is sufficient for a mini gel system. For a larger chamber, make 1 L of 1x buffer.
# Remove strip from the bottom of the gel to expose it.
# Secure the gel, exposed side facing the outer wall, inside the chamber. Note: If running a single gel, set up a dummy gel mold opposite the actual gel.
# Use the 1x running buffer to fill the inner camber to the top (above the gel lanes). For a mini gel system, this will take about 200 mL.
# '''IMPORTANT''': If using NuPAGE buffer, add 500 μL Antioxidant into the buffer in the inner chamber drop-wise, evenly over the surface of the buffer.
# Next, fill the outer chamber just so that the bottom of the gel is submerged.
# Gently pull out the well comb and discard it.
# Carefully load the ladder, samples, and dummy samples using flexible skinny pipette tips.
# Run the gel @ 120 V until the dye front reaches the very bottom.




'''HELPFUL RESOURCES'''
'''HELPFUL RESOURCES'''
* Gel setup: Video - [https://www.youtube.com/watch?v=wWFZ_Pf-hf4 Running the Gel on the XCell SureLock® system]
* Electroblotting wet transfer: Video - [https://www.youtube.com/watch?v=uTY96pBj26o How to perform a traditional wet protein transfer using the XCell SureLock® Blot Module]




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Revision as of 15:09, 24 March 2015

<- Back to Protocols

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
  • 10x PBS
  • Tween-20 (e.g., G-Biosciences 786-517)


EQUIPMENT

  • Electroblotting chamber (e.g. Invitorgen XCell SureLock)


PROCEDURE

Prepare 1x Transfer Buffer [12.5 mM Tris; 96 mM glycine; 10% methanol]

  1. Add ~200 mL dH2O, 25 mL Tris-glycine concentrate, and 50 mL methanol to a 500 mL bottle. Swirl to mix.
  2. Fill up to 500 mL with dH2O (+ ~225 mL dH2O). Note: deionized water from the tap is sufficient.

Prepare 1x PBS-tween [? mM Na2HPO4; ? mM NaH2PO4; ? nmM NaCl; 0.1% Tween-20; pH ~7.2]

  1. Add ~200 mL dH2O, 50 mL 10x PBS, and 0.5 mL Tween-20 to a 500 mL bottle. Swirl to mix.
  2. 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

  1. Remove the gel from the mold, trim the top and bottom, and soak in Western blot transfer buffer “back” side up.


Prepare the electrophoresis chamber


HELPFUL RESOURCES


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