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=SDS-PAGE=
=SDS-PAGE=
by Karmella Haynes, 2015<br><br>
by Karmella Haynes, 2015<br><br>
Principle: Proteins are denatured and given a negative charge with a detergent (SDS), loaded to the top of a vertical gel, then separated by protein fragment size by applying an electric charge
Principle: Proteins are denatured and given a negative charge with a detergent (SDS), disulfide bonds are broken with a redox agent, samples are loaded to the top of a vertical gel, then separated by protein fragment size by applying an electric charge.




'''MATERIALS'''
'''MATERIALS'''
* Protein sample buffer (e.g. NuPAGE® LDS Sample Buffer 4X)
* Protein sample buffer (e.g. NuPAGE® LDS Sample Buffer 4X)
* 1M DL-Dithiothreitol (DTT) redox agent (Sigma D0632-1G)
* Redox agent - 1M DL-Dithiothreitol (DTT) (Sigma D0632-1G)
* Antioxidant - NuPAGE® Antioxidant (Life Technologies NP0005)
* Polyacrylamide gel (use the appropriate gel for your application)
* Polyacrylamide gel (use the appropriate gel for your application)
** Bis-Tris - small to mid-size proteins
** Bis-Tris - small to mid-size proteins
** Tris-Acetate - large proteins
** Tris-Acetate - large proteins
** Tris-Glycine - "stacked" gel for running large and small proteins on the same gel
* Running buffer (use the appropriate buffer for your gel)
* Running buffer (use the appropriate buffer for your gel)
** Bis-Tris gel - MES SDS or MOPS SDS (e.g., 20x NuPAGE® MOPS SDS Running Buffer, Life Technologies NP0001)
** Bis-Tris gel - MES SDS or MOPS SDS (e.g., 20x NuPAGE® MOPS SDS Running Buffer, Life Technologies NP0001)
** Tris-Acetate gel - Tris-Acetate SDS
** Tris-Acetate gel - Tris-Acetate SDS
** Tris-Glycine gel - Tris-Glycine buffer (e.g., 20x Novex® Tris-Glycine SDS Running Buffer, Life Technologies)
* Antioxidant - Only if using NuPAGE buffer, NuPAGE® Antioxidant (Life Technologies NP0005)




EQUIPMENT
'''EQUIPMENT'''
* Vertical gel electrophoresis chamber (e.g. Mini protean Bio-Rad)
* Vertical gel electrophoresis chamber (e.g. Invitrogen XCell SureLock)
** Note: make sure the gel cassette is compatible with the electrophoresis system




PROCEDURE
PROCEDURE
'''Prepare protein samples'''
# The final volume of each loaded sample is typically 20 μL. Check the specifications of the gel for well capacity.
# The final volume of each loaded sample is typically 20 μL. Check the specifications of the gel for well capacity.
# Prepare Sample buffer+DDT: dilute DTT in the concentrated loading dye so that the final 1x concentration will be 50 mM. Make enough of this solution for the total number of samples.
# 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: For a 4x sample buffer, make 200 mM DTT in sample buffer, (1.0 μL DTT + 4.0 μL sample buffer) x total number of samples
## Example 1: 20 μL final loading volume - (5.0 μL 4x sample buffer + 1.0 μL 1M DTT) = 14.0 μL protein
# Prepare protein samples: If needed, make dilutions of the protein samples in separate tubes. Make sure the protein sample volume = the final volume - concentrated sample buffer volume
## Example 1: 20 μL final loading volume - (10.0 μL 2x sample buffer + 1.0 μL 1M DTT) = 9.0 μL protein
## Example: For a 4x sample buffer, each ready-to-laod sample will have 5.0 sample buffer+DTT, and 14.0
# Make a loading buffer master mix: multiplier = total number of lanes + 1 for pipetting error. Example: if using one gel with 12 lanes, the multiplier is 13
3. Add 2X loading dye+DTT equal to the volume of each sample.  
## '''Example - for a 12-lane gel, using 4x sample buffer''': 5.0 μL 4x sample buffer * 13 = 65. 1.0 μL 1M DTT * 13 = 13. Add '''65 μL 4x sample buffer + 13 μL 1M DTT into a 1.5 mL tube'''
Note: If no. of samples, including protein ladder, is fewer than the no. of lanes in the gel, make dummy samples of 1X Loading dye+DTT. Dummy samples allow proteins to migrate correctly.
# Set up the protein samples: in fresh 0.5 mL tubes, add protein and sample buffer master mix.
4. Heat samples, excluding the ladder, @ 100°C/ 5 min.
## '''Example - for a 12-lane gel, using 4x sample buffer''': Add '''6.0 μL sample buffer master mix + 14.0 μL protein''' per sample.  
5. Set up the pre-cast acrylamide gel in the electrophoresis chamber: Pull out comb from top and remove strip from bottom. If running a single gel, set up a dummy gel mold opposite the gel. Fill the inner camber to the top (above the lanes) and the outer chamber just so that the bottom of the gel is submerged.
# If running fewer samples than the total number of lanes, make "dummy" samples: for instance, 6.0 μL of master mix + 14.0 μL water for each empty lane
6. Carefully load the ladder, samples, and dummy samples using flexible skinny pipette tips.
# Heat samples, '''excluding the protein standard(s)''', @ 100°C/ 5 min. Cool at room temp.
7. Run the gel @ 120 V until the dye front reaches the very bottom.


'''Prepare the gel and 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.


'''Table 1. Standard sample set-up'''
{| {{table}}
|-
| Reagent || Tube 1<br>(0 μg BSA) || Tube 2<br>(1 μg BSA) || Tube 3<br>(2 μg BSA) || Tube 4<br>(4 μg BSA) || Tube 5 <br>8 μg BSA) || Tube 5<br>(16 μg BSA)
|-
| Bradford Reagent || 500 μL || 500 μL || 500 μL || 500 μL || 500 μL || 500 μL
|-
| BSA diluted to 1 μg/μL || 0 μL || 1.0 μL || 2.0 μL || 4.0 μL || 8.0 μL || 16.0 μL
|}


'''HELPFUL RESOURCES'''
* Gel setup: Video - [https://www.youtube.com/watch?v=wWFZ_Pf-hf4 Running the Gel on the XCell SureLock® system]


What to do with your data: calculate unknown protein concentration(s)
# Subtract the blank OD 590 value (Tube 1) from all other values.
# Plot a standard curve (using Excel) with BSA concentration (x-axis) vs. Absorbance at 590 nm (y-axis). See [http://openwetware.org/wiki/Image:KAH_012510_chart1.tif this example].
# Add a '''line''' of best fit (not a curve) and display the equation.
# Solve the equation for ''x''. Substitute ''y'' with the background-subtracted OD 590 for the unknowns. The x value will be the protein concentration of the unknown as μg/'''5.0 μL''' (because you used 5 μL to set up the assay sample).
# Convert the unknowns to μg/μL: (x μg/5.0 μL) / 5 = x μg/μL


</div>
</div>

Latest revision as of 16:16, 12 August 2015

<- Back to Protocols

SDS-PAGE

by Karmella Haynes, 2015

Principle: Proteins are denatured and given a negative charge with a detergent (SDS), disulfide bonds are broken with a redox agent, samples are loaded to the top of a vertical gel, then separated by protein fragment size by applying an electric charge.


MATERIALS

  • Protein sample buffer (e.g. NuPAGE® LDS Sample Buffer 4X)
  • Redox agent - 1M DL-Dithiothreitol (DTT) (Sigma D0632-1G)
  • Polyacrylamide gel (use the appropriate gel for your application)
    • Bis-Tris - small to mid-size proteins
    • Tris-Acetate - large proteins
    • Tris-Glycine - "stacked" gel for running large and small proteins on the same gel
  • Running buffer (use the appropriate buffer for your gel)
    • Bis-Tris gel - MES SDS or MOPS SDS (e.g., 20x NuPAGE® MOPS SDS Running Buffer, Life Technologies NP0001)
    • Tris-Acetate gel - Tris-Acetate SDS
    • Tris-Glycine gel - Tris-Glycine buffer (e.g., 20x Novex® Tris-Glycine SDS Running Buffer, Life Technologies)
  • Antioxidant - Only if using NuPAGE buffer, NuPAGE® Antioxidant (Life Technologies NP0005)


EQUIPMENT

  • Vertical gel electrophoresis chamber (e.g. Invitrogen XCell SureLock)
    • Note: make sure the gel cassette is compatible with the electrophoresis system


PROCEDURE

Prepare protein samples

  1. The final volume of each loaded sample is typically 20 μL. Check the specifications of the gel for well capacity.
  2. 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)
    1. Example 1: 20 μL final loading volume - (5.0 μL 4x sample buffer + 1.0 μL 1M DTT) = 14.0 μL protein
    2. Example 1: 20 μL final loading volume - (10.0 μL 2x sample buffer + 1.0 μL 1M DTT) = 9.0 μL protein
  3. Make a loading buffer master mix: multiplier = total number of lanes + 1 for pipetting error. Example: if using one gel with 12 lanes, the multiplier is 13
    1. Example - for a 12-lane gel, using 4x sample buffer: 5.0 μL 4x sample buffer * 13 = 65. 1.0 μL 1M DTT * 13 = 13. Add 65 μL 4x sample buffer + 13 μL 1M DTT into a 1.5 mL tube
  4. Set up the protein samples: in fresh 0.5 mL tubes, add protein and sample buffer master mix.
    1. Example - for a 12-lane gel, using 4x sample buffer: Add 6.0 μL sample buffer master mix + 14.0 μL protein per sample.
  5. If running fewer samples than the total number of lanes, make "dummy" samples: for instance, 6.0 μL of master mix + 14.0 μL water for each empty lane
  6. Heat samples, excluding the protein standard(s), @ 100°C/ 5 min. Cool at room temp.

Prepare the gel and electrophoresis chamber

  1. Prepare the running buffer: dilute the buffer to make 500 mL of 1x running buffer.
    1. Note: 500 mL is sufficient for a mini gel system. For a larger chamber, make 1 L of 1x buffer.
  2. Remove strip from the bottom of the gel to expose it.
  3. 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.
  4. 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.
  5. 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.
  6. Next, fill the outer chamber just so that the bottom of the gel is submerged.
  7. Gently pull out the well comb and discard it.
  8. Carefully load the ladder, samples, and dummy samples using flexible skinny pipette tips.
  9. Run the gel @ 120 V until the dye front reaches the very bottom.


HELPFUL RESOURCES


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