Melbourne/Preparing Protein Samples for SDS PAGEl

From 2007.igem.org

< Melbourne(Difference between revisions)
 
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====Method from primary and secondary reagents====
====Method from primary and secondary reagents====
=====Primary & secondary Reagents Required including controls=====
=====Primary & secondary Reagents Required including controls=====
-
*[[Melbourne/Preparing a poly-acrylamide gel|polyacrylamide gel]]
+
*[[Melbourne/Secondary Reagent LB|LB]]
-
*1X SDS buffer (6.05g [[Melbourne/primary Tris|Tris]], 28.83g glycine, 2g SDS per liter)
+
*Selective Antibiotic
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*Benchmark Protein Ladder
+
*Bacterial Colony containing inducible protein plasmid
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*Denatured protein samples
+
*[[Melbourne/primary IPTG|IPTG]]
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*Commassie blue (50% methanol, 10% acetic acid, 0.2% commassie)
+
*PBS
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*Destain solution (10% methanol, 10% acetic acid)
+
*Triton-X 1000 (or alternative non-ionic detergent)
 +
*SDS sample buffer
 +
*PMSF
=====Method including controls=====
=====Method including controls=====
#Grow up overnight culture of bacteria to be induced.
#Grow up overnight culture of bacteria to be induced.
#Pipette 1ml of culture into a conical flask containing 100ml of LB and selective antibiotic.
#Pipette 1ml of culture into a conical flask containing 100ml of LB and selective antibiotic.
-
#Culture the bacteria at 25 degrees C till the optical density at 600nm reaches about 1.
+
#Culture the bacteria at 25 (or 37) degrees C with shaking till the optical density at 600nm reaches about 1.
-
#Add IPTG to a final concentration of 1mM.
+
#Add IPTG to a final concentration of 100uM.
 +
#Continue to culture the bacteria for 2-4 hours (depending on the protein).
 +
#Centrifuge at about 8000g for 10 minutes.
 +
# Discard the supernatant. Place on ice.
 +
#Resuspend using a pipette in 5ml of cold 1 X PBS (or TBS)
 +
#Add 5ul of PMSF
 +
# Sonicate 2 or 3 times in 15 second bursts with about 2 minutes rest in between bursts (keep the tube on ice at all times- especially between bursts).
 +
# Add Triton X-100 to a final volume of 1%. Mix gently for 30 minutes e.g. on a rocker.
 +
#Centrifuge for 10 minutes at 12,000g.
 +
#Save both the supernatant and the pellet.<BR>-Transfer the supernatant to a new tube. This is the soluble fraction.<BR>-Resuspend the pellet in 1ml of 1XPBS. This is the insoluble fraction.<BR>Both fractions can be frozen at this point if down-stream applications involve denaturing the protein (e.g. using SDS).
 +
#To 1ml of the above fractions (i.e. 1ml from the supernatant and all of the pellet re-suspention) add SDS sample (loading) buffer to make 1X buffer.
 +
# Heat at 100 degrees for 10 minutes. This is your protein sample ready to load on SDS-PAGE. They can be safely stored in the freezer.
-
1. Centrifuge at about 8000g=8000rpm for 10 minutes.
 
-
2. Discard the supernatant. Place on ice.
 
-
3. Resuspend using a pipette in 5ml of cold 1 X PBS (or TBS)
 
-
4. Sonicate 2 or 3 times in 15 second bursts with about 2 minutes rest
 
-
in between bursts (keep the tube on ice at all times- especially
 
-
between bursts).
 
-
 
-
5. Add Triton X-100 (this can be changed to a different detergent
 
-
depending on what you want to solubilize- but I suggest you try this
 
-
first) to a final volume of 1%. Mix gently for 30 minutes on a wheel
 
-
or rocker or whatever is available.
 
-
 
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6. Centrifuge for 10 minutes at 10,000rpm=12,000g.
 
-
7. Save both the supernatant and the pellet.
 
-
-Transfer the supernatant to a new tube. This is the soluble fraction.
 
-
-Resuspend the pellet in 1ml of 1XPBS. This is the insoluble fraction.
 
-
 
-
Both fractions can be frozen at this point if down-stream applications
 
-
involve denaturing the protein (e.g. using SDS as is in this case).
 
-
 
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8. To 1ml of the above fractions (i.e. 1ml from the supernatant and
 
-
all of the pellet re-suspention) add SDS sample (loading) buffer to
 
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make 1Xbuffer. This is a blue buffer that you should ask for in the
 
-
lab. The buffer is usually 6X in the original form so add 200
 
-
microliters of it to your fractions.
 
-
 
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9. Heat at 100 degrees for 10 minutes. This is your protein sample
 
-
ready to load on SDS-PAGE. They can be safely stored in the freezer.
 
-
 
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10. Optional. You can do a Bradford assay to quantitate the amount to
 
-
protein you have in each sample to equalize the amount of protein in
 
-
each lane. Personally for what you are doing now I don't think this is
 
-
necessary. I advise to just add 10 microliters of each sample to the
 
-
gel.
 
=====Equipement Required=====
=====Equipement Required=====
*Pipette and tips
*Pipette and tips
-
*SDS PAGE apparatus
+
*Conical flask
-
*Power source
+
*Spectrophotometer
 +
*Plastic Cuvettes
 +
*Shaker
 +
*Centrifuge
 +
*[[Melbourne/primary ice|ice]]
 +
*Sonicator
 +
*Heat block
=====References=====
=====References=====
*
*

Latest revision as of 13:00, 29 September 2007

<Return to list of protocols> <Team home page>

  • Applications:
  • Inducing bacteria to produce specific proteins
  • Time to complete protocol:
    • Lab time: 15min, 5min, 5min
    • Waiting time:2h, 1h, 30min
  • Approximate cost of materials: $

Contents

Method from primary and secondary reagents

Primary & secondary Reagents Required including controls
  • LB
  • Selective Antibiotic
  • Bacterial Colony containing inducible protein plasmid
  • IPTG
  • PBS
  • Triton-X 1000 (or alternative non-ionic detergent)
  • SDS sample buffer
  • PMSF
Method including controls
  1. Grow up overnight culture of bacteria to be induced.
  2. Pipette 1ml of culture into a conical flask containing 100ml of LB and selective antibiotic.
  3. Culture the bacteria at 25 (or 37) degrees C with shaking till the optical density at 600nm reaches about 1.
  4. Add IPTG to a final concentration of 100uM.
  5. Continue to culture the bacteria for 2-4 hours (depending on the protein).
  6. Centrifuge at about 8000g for 10 minutes.
  7. Discard the supernatant. Place on ice.
  8. Resuspend using a pipette in 5ml of cold 1 X PBS (or TBS)
  9. Add 5ul of PMSF
  10. Sonicate 2 or 3 times in 15 second bursts with about 2 minutes rest in between bursts (keep the tube on ice at all times- especially between bursts).
  11. Add Triton X-100 to a final volume of 1%. Mix gently for 30 minutes e.g. on a rocker.
  12. Centrifuge for 10 minutes at 12,000g.
  13. Save both the supernatant and the pellet.
    -Transfer the supernatant to a new tube. This is the soluble fraction.
    -Resuspend the pellet in 1ml of 1XPBS. This is the insoluble fraction.
    Both fractions can be frozen at this point if down-stream applications involve denaturing the protein (e.g. using SDS).
  14. To 1ml of the above fractions (i.e. 1ml from the supernatant and all of the pellet re-suspention) add SDS sample (loading) buffer to make 1X buffer.
  15. Heat at 100 degrees for 10 minutes. This is your protein sample ready to load on SDS-PAGE. They can be safely stored in the freezer.
Equipement Required
  • Pipette and tips
  • Conical flask
  • Spectrophotometer
  • Plastic Cuvettes
  • Shaker
  • Centrifuge
  • ice
  • Sonicator
  • Heat block
References
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