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Jacobs:Protocol NuPAGE Electrophoresis and Western Blotting for Proteins: Difference between revisions
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Jacobs:Protocol NuPAGE Electrophoresis and Western Blotting for Proteins (view source)
Revision as of 09:27, 12 November 2009
, 12 November 2009→Procedure
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#Turn on heating block to 70°C about 15 minutes before step 6. | #Turn on heating block to 70°C about 15 minutes before step 6. | ||
#Prepare 1000 ml of 1x NuPAGE® SDS Running Buffer using NuPAGE® SDS Running Buffer(20X) as follows in 1000mL Pyrex Bottle and mix thoroughly, Running Buffer: | #Prepare 1000 ml of 1x NuPAGE® SDS Running Buffer using NuPAGE® SDS Running Buffer(20X) as follows in 1000mL Pyrex Bottle and mix thoroughly, Running Buffer: | ||
##NuPAGE® SDS Running Buffer (20X MOPS) 50 ml | |||
##Deionized Water 950 ml | |||
##Total Volume 1000 ml | |||
##Set aside 800 ml of the 1X NuPAGE® SDS Running Buffer for use in the lower (Outer) Buffer Chamber of the XCell SureLock Mini-Cell | |||
##200 ml of 1X NuPAGE® SDS Running for use in the Upper (Inner) Buffer Chamber of the XCell SureLock. Mini-Cell. | |||
##Keep at room temperature | |||
#Prepare reducing sample in this order (Total volume depends on strength of protein): | #Prepare reducing sample in this order (Total volume depends on strength of protein): | ||
##Sample x μl | |||
##NuPAGE LDS Sample Buffer (4X) 4°C | |||
##NuPAGE® Reducing Agent (10X) 4°C | |||
##Maximum Total Volume 25 μl | |||
##Notes: For reduced sample, add the reducing agent immediately prior to electrophoresis to obtain the best results. The presence of more glycerol also increases the viscosity of the NuPAGE® LDS Sample Buffer. By bringing the NuPAGE® LDS Sample Buffer to room temperature (25°C), the buffer is more manageable. | |||
# Heat the sample for denaturing electrophoresis (reduced or non-reduced) at 70°C for 10 minutes for optimal results. | # Heat the sample for denaturing electrophoresis (reduced or non-reduced) at 70°C for 10 minutes for optimal results. | ||
# Remove the NuPAGE® Gel from the pouch | # Remove the NuPAGE® Gel from the pouch | ||
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'''Western Blotting ''' | '''Western Blotting ''' | ||
# Prepare 1000 ml of 1X NuPAGE® Transfer Buffer using the NuPAGE® Transfer Buffer (20X) as follows in 1000mL Pyrex bottle: Transfer Buffer: | # Prepare 1000 ml of 1X NuPAGE® Transfer Buffer using the NuPAGE® Transfer Buffer (20X) as follows in 1000mL Pyrex bottle: Transfer Buffer: | ||
##NuPAGE® Transfer Buffer (20X) 50 ml at room temp | |||
##Methanol (100 mL for 1 gel) (200mL for 2 gels) | |||
##Deionized Water (849 mL for 1 gel) (749 mL for 2 gels) | |||
##Total Volume 1000 ml | |||
##Put in 4°C | |||
# Use about 700 ml of 1X NuPAGE® Transfer Buffer to soak the pads until saturated during electrophoresis. Remove the air bubbles by squeezing the pads while they are submerged in buffer. Removing the air bubbles is essential as they can block the transfer of biomolecules if they are not removed. | # Use about 700 ml of 1X NuPAGE® Transfer Buffer to soak the pads until saturated during electrophoresis. Remove the air bubbles by squeezing the pads while they are submerged in buffer. Removing the air bubbles is essential as they can block the transfer of biomolecules if they are not removed. | ||
# Put 650mL dH20 into 4°C for step 21. | # Put 650mL dH20 into 4°C for step 21. | ||
