NanoBio: Protein Gels

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(New page: ==Protein Gels== ===Materials=== *Protein loading dye (we have 4X) *Beta mercaptoethanol (BME, a reducing agent) *Protein ladder *Samples *Protein gel ===Selecting the right gel=== *There ...)
Line 10: Line 10:
===Preparing your samples===
===Preparing your samples===
#Normalize the protein concentrations. So if you know Sample A is a more dilute than Sample B, you can mix some B with water to normalize it to the lower concentration sample.
#Normalize the protein concentrations. So if you know Sample A is a more dilute than Sample B, you can mix some B with water to normalize it to the lower concentration sample.
-
##Each well holds ~50uL maximum. You can aim for 40uL load.
+
#*Each well holds ~50uL maximum. You can aim for 40uL load.
#Mix up enough dye with 1/10 BME for all of your samples.
#Mix up enough dye with 1/10 BME for all of your samples.
-
##Example: Your dye is 4X. You'll need 40/4 = 10uL for each sample. So mix up 9uL dye + 1uL BME.
+
#*Example: Your dye is 4X. You'll need 40/4 = 10uL for each sample. So mix up 9uL dye + 1uL BME.
#Add Dye+BME mix to your samples.
#Add Dye+BME mix to your samples.
#Heat samples to 95 C for 5 minutes.
#Heat samples to 95 C for 5 minutes.
-
##Note: Tube clamps are suggested since the air might expand. Don't be alarmed if they pop on you!
+
#*Note: Tube clamps are suggested since the air might expand. Don't be alarmed if they pop on you!
-
##This process breaks disulfide bonds and more or less linearizes the proteins so shape doesn't affect running.
+
#*This process breaks disulfide bonds and more or less linearizes the proteins so shape doesn't affect running.
#Pulse centrifuge to collect condensation.
#Pulse centrifuge to collect condensation.
===Load and run===
===Load and run===
 +
#Open up the gel box and stick your gel in. It holds up to two gels.
 +
#Add running buffer to the 2 cavities at the ends.
 +
#Load samples. If you are not using all the wells, you may want to add loading buffer and water (~30uL total volume) to bordering lanes. This will reduce gel "smiling".
 +
#*Ensure that you load ladder and samples in a fashion so you can track top/bottom and left/right!
 +
#Run at 200V for 1hr.
 +
===Staining (using Imperial stain)===
 +
#Add water to cover the gel and shake on an orbital shaker for 5 minutes.
 +
#Shake the stain bottle
 +
#Cover the gel with stain.
 +
*Option 1: Microwave (quick but not as dark) 6-12ng sensitivity
 +
*#Place gel into a microwave-safe container and microwave for up to a minute.
 +
*#*Don't boil it. If it boils, just let it sit at just preboil temperature for the remainder of the time.
 +
*#Pour out dye (you can save it and reuse it if you store it in a dark/Al-foil'd container)
 +
*#Add water and microwave for a minute.
 +
*Option 2: Overnight (tried and true) up to 3ng sensitivity
 +
*#Staining and washing are done with the orbital shaker. Use diH2O for washing.
 +
{|
 +
|-
 +
!Resolution
 +
!Stain
 +
!Wash
 +
|-
 +
|<3ng
 +
|2 hours
 +
|overnight
 +
|-
 +
|3-6ng
 +
|1 hour
 +
|1-2 hours
 +
|-
 +
|6-12ng
 +
|5-10 min
 +
|15 minutes
 +
|-
 +
|}
 +
Source: [http://www.ohsu.edu/proteomics/links/pdfs/PSR_Protein_Gels.pdf PSR Protein Gels]
 +
 +
[[User:Arthur K Yu|Arthur K Yu]] 20:44, 8 July 2008 (UTC)

Revision as of 16:44, 8 July 2008

Contents

Protein Gels

Materials

  • Protein loading dye (we have 4X)
  • Beta mercaptoethanol (BME, a reducing agent)
  • Protein ladder
  • Samples
  • Protein gel

Selecting the right gel

  • There is a chart in the room with the gel running boxes that shows the different gel types and the resolution provided by each. Choose an appropriate gel so you can see the proteins you want.

Preparing your samples

  1. Normalize the protein concentrations. So if you know Sample A is a more dilute than Sample B, you can mix some B with water to normalize it to the lower concentration sample.
    • Each well holds ~50uL maximum. You can aim for 40uL load.
  2. Mix up enough dye with 1/10 BME for all of your samples.
    • Example: Your dye is 4X. You'll need 40/4 = 10uL for each sample. So mix up 9uL dye + 1uL BME.
  3. Add Dye+BME mix to your samples.
  4. Heat samples to 95 C for 5 minutes.
    • Note: Tube clamps are suggested since the air might expand. Don't be alarmed if they pop on you!
    • This process breaks disulfide bonds and more or less linearizes the proteins so shape doesn't affect running.
  5. Pulse centrifuge to collect condensation.

Load and run

  1. Open up the gel box and stick your gel in. It holds up to two gels.
  2. Add running buffer to the 2 cavities at the ends.
  3. Load samples. If you are not using all the wells, you may want to add loading buffer and water (~30uL total volume) to bordering lanes. This will reduce gel "smiling".
    • Ensure that you load ladder and samples in a fashion so you can track top/bottom and left/right!
  4. Run at 200V for 1hr.

Staining (using Imperial stain)

  1. Add water to cover the gel and shake on an orbital shaker for 5 minutes.
  2. Shake the stain bottle
  3. Cover the gel with stain.
  • Option 1: Microwave (quick but not as dark) 6-12ng sensitivity
    1. Place gel into a microwave-safe container and microwave for up to a minute.
      • Don't boil it. If it boils, just let it sit at just preboil temperature for the remainder of the time.
    2. Pour out dye (you can save it and reuse it if you store it in a dark/Al-foil'd container)
    3. Add water and microwave for a minute.
  • Option 2: Overnight (tried and true) up to 3ng sensitivity
    1. Staining and washing are done with the orbital shaker. Use diH2O for washing.
Resolution Stain Wash
<3ng 2 hours overnight
3-6ng 1 hour 1-2 hours
6-12ng 5-10 min 15 minutes

Source: PSR Protein Gels

Arthur K Yu 20:44, 8 July 2008 (UTC)

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