Knight:NuPAGE electrophoresis/Slow staining: Difference between revisions
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*[http://www.invitrogen.com/content.cfm?pageid=9869 Invitrogen SimplyBlue SafeStain] | *[http://www.invitrogen.com/content.cfm?pageid=9869 Invitrogen SimplyBlue SafeStain] | ||
*Plastic tray | *Plastic tray | ||
**''Use lid of a 1000μL pipette tip box.'' | |||
*Orbital shaker | *Orbital shaker | ||
*Kimwipes | *Kimwipes | ||
| Line 14: | Line 15: | ||
#Discard water. | #Discard water. | ||
#Repeat steps 1-3 two more times. | #Repeat steps 1-3 two more times. | ||
#Add 20mL SimplyBlue SafeStain. | #Add 20mL SimplyBlue SafeStain (enough to just cover the gel). | ||
#Shake staining tray for 1 hr on orbital shaker at room temperature. | #Shake staining tray for 1 hr on orbital shaker at room temperature. | ||
#*''Overnight is better for improved sensitivity.'' | #*''Overnight is better for improved sensitivity.'' | ||
| Line 21: | Line 22: | ||
#*''If the gel has been stained overnight, let it destain for a few hours.'' | #*''If the gel has been stained overnight, let it destain for a few hours.'' | ||
This protocol worked less well for me. However, I learned later that the typical trick people use is to include a kimwipe in the destain step to help soak up the stain. This will likely improve the signal to noise on the gel. | ==Notes== | ||
*This protocol worked less well for me. However, I learned later that the typical trick people use is to include a kimwipe in the destain step to help soak up the stain. This will likely improve the signal to noise on the gel. | |||
*To date, the best staining tray I've found is the lid of a 1000μL pipette tip box. Then use a piece of mesh that just fits inside the lide to either keep the gel in place while changing solutions or to move the gel to and from the light box. This method requires smaller volumes of stain than the staining tray from Invitrogen. | |||
[[Category:Protein]] [[Category:In vitro]] [[Category:Protocol]] | |||
Latest revision as of 09:24, 23 August 2007
Overview
A slow protocol for visualizing bands on a polyacrylamide gel via coomassie-like staining.
Materials
- Deionized water
- Invitrogen SimplyBlue SafeStain
- Plastic tray
- Use lid of a 1000μL pipette tip box.
- Orbital shaker
- Kimwipes
Procedure
- Add 100mL deionized water in staining tray.
- Shake staining tray for 5 min on orbital shaker at room temperature.
- Discard water.
- Repeat steps 1-3 two more times.
- Add 20mL SimplyBlue SafeStain (enough to just cover the gel).
- Shake staining tray for 1 hr on orbital shaker at room temperature.
- Overnight is better for improved sensitivity.
- Add 100mL deionized water.
- Shake staining tray for 1 hr or more on orbital shaker at room temperature.
- If the gel has been stained overnight, let it destain for a few hours.
Notes
- This protocol worked less well for me. However, I learned later that the typical trick people use is to include a kimwipe in the destain step to help soak up the stain. This will likely improve the signal to noise on the gel.
- To date, the best staining tray I've found is the lid of a 1000μL pipette tip box. Then use a piece of mesh that just fits inside the lide to either keep the gel in place while changing solutions or to move the gel to and from the light box. This method requires smaller volumes of stain than the staining tray from Invitrogen.
