Haynes:PAGE: Difference between revisions

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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), 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) )
'''MATERIALS'''
* DL-Dithiothreitol (DTT) redox agent (Sigma D0632-1G)
* Protein sample buffer (e.g. NuPAGE® LDS Sample Buffer 4X)
* SDS Running buffer - 20x NuPAGE® MOPS SDS Running Buffer (Life Technologies NP0001)
* 1M DL-Dithiothreitol (DTT) redox agent (Sigma D0632-1G)
* Antioxidant - NuPAGE® Antioxidant (Life Technologies NP0005)
* 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
* 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




EQUIPMENT
EQUIPMENT
* Vertical gel electrphoresis chamber (e.g.  
* Vertical gel electrophoresis chamber (e.g. Mini protean Bio-Rad)




PROCEDURE
PROCEDURE
# Label enough 1.5 mL eppendorf tubes for one blank (1) , five standard samples (2-6), and all of your unknown samples (7-...''n'').
# The final volume of each loaded sample is typically 20 μL. Check the specifications of the gel for well capacity.
# Add 500 μL Bradford Reagent to each tube. You will add protein to these later, and ignore the negligible change caused by the additional volume.
# 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.
# Dilute the stock BSA in a new tube to make 50 μL of 1 μg/μL BSA. Example: if the stock BSA is 10 mg/mL, add 5 μL of BSA to 45 μL dH<sub>2</sub>O in a fresh tube.
## 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
# Add a BSA standard protein solution to tubes 1-6. See '''Table 1'''.
# 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
# Add 5.0 μL of unknown to each remaining tube. Keep track of your samples with good labeling!
## Example: For a 4x sample buffer, each ready-to-laod sample will have 5.0 sample buffer+DTT, and 14.0
# Close all caps and invert the tubes to thoroughly mix the samples.
3. Add 2X loading dye+DTT equal to the volume of each sample.
# Transfer 200 μL of the blank (tube one) into the first well in a clear 96-well flat-bottom plate.
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.
# Do the same for the other samples, using new wells.
4. Heat samples, excluding the ladder, @ 100°C/ 5 min.
# Use a plate reader to record absorbance at 590 nm (OD 590). If using the BioTek Synergy H1 Software, set up a new protocol and under Procedure > Action > Read use the following settings
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.
## Detection Method = Absorbance
6. Carefully load the ladder, samples, and dummy samples using flexible skinny pipette tips.
## Read Type = Endpoint
7. Run the gel @ 120 V until the dye front reaches the very bottom.
## Wavelength (1) = 590 nm (type-in the value manually)




Revision as of 18:12, 19 March 2015

<- Back to Protocols

SDS-PAGE

by Karmella Haynes, 2015

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


MATERIALS

  • Protein sample buffer (e.g. NuPAGE® LDS Sample Buffer 4X)
  • 1M DL-Dithiothreitol (DTT) redox agent (Sigma D0632-1G)
  • Antioxidant - NuPAGE® Antioxidant (Life Technologies NP0005)
  • Polyacrylamide gel (use the appropriate gel for your application)
    • Bis-Tris - small to mid-size proteins
    • Tris-Acetate - large proteins
  • 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


EQUIPMENT

  • Vertical gel electrophoresis chamber (e.g. Mini protean Bio-Rad)


PROCEDURE

  1. The final volume of each loaded sample is typically 20 μL. Check the specifications of the gel for well capacity.
  2. 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.
    1. 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
  3. 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
    1. Example: For a 4x sample buffer, each ready-to-laod sample will have 5.0 sample buffer+DTT, and 14.0

3. Add 2X loading dye+DTT equal to the volume of each sample. 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. 4. Heat samples, excluding the ladder, @ 100°C/ 5 min. 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. 6. Carefully load the ladder, samples, and dummy samples using flexible skinny pipette tips. 7. Run the gel @ 120 V until the dye front reaches the very bottom.


Table 1. Standard sample set-up

Reagent Tube 1
(0 μg BSA)		 Tube 2
(1 μg BSA)		 Tube 3
(2 μg BSA)		 Tube 4
(4 μg BSA)		 Tube 5
8 μg BSA)		 Tube 5
(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


What to do with your data: calculate unknown protein concentration(s)

  1. Subtract the blank OD 590 value (Tube 1) from all other values.
  2. Plot a standard curve (using Excel) with BSA concentration (x-axis) vs. Absorbance at 590 nm (y-axis). See this example.
  3. Add a line of best fit (not a curve) and display the equation.
  4. 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).
  5. Convert the unknowns to μg/μL: (x μg/5.0 μL) / 5 = x μg/μL
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