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 09:25, 12 November 2009

No change in size ,  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:  
1. NuPAGE® SDS Running Buffer (20X MOPS) 50 ml  
*NuPAGE® SDS Running Buffer (20X MOPS) 50 ml  
2. Deionized Water 950 ml  
*Deionized Water 950 ml  
3. Total Volume 1000 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  
*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.  
*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
*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):
1. Sample x μl  
*Sample x μl  
2. NuPAGE LDS Sample Buffer (4X) 4°C
*NuPAGE LDS Sample Buffer (4X) 4°C
3. NuPAGE® Reducing Agent (10X) 4°C  
*NuPAGE® Reducing Agent (10X) 4°C  
4. Maximum Total Volume 25 μl  
*Maximum Total Volume 25 μl  
5. 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.  
*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:  
1. NuPAGE® Transfer Buffer (20X) 50 ml at room temp  
*NuPAGE® Transfer Buffer (20X) 50 ml at room temp  
2. Methanol (100 mL for 1 gel)  (200mL for 2 gels)
*Methanol (100 mL for 1 gel)  (200mL for 2 gels)
3. Deionized Water (849 mL for 1 gel)  (749 mL for 2 gels)  
*Deionized Water (849 mL for 1 gel)  (749 mL for 2 gels)  
4. Total Volume 1000 ml  
*Total Volume 1000 ml  
5. Put in 4°C
*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.
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# Rinse membrane twice with dwater  
# Rinse membrane twice with dwater  
# Block membrane 20min-2hours (depending on protein) at room temperature with agitation in freshly prepared 5% albumin (Catalog 20-200) in TBS (TBS-MLK)  
# Block membrane 20min-2hours (depending on protein) at room temperature with agitation in freshly prepared 5% albumin (Catalog 20-200) in TBS (TBS-MLK)  
# Incubate membrane overnight at 4°C with agitation and anti-x, diluted 1:2000 in freshly prepared TBS-MLK (dilution depends on protein)
# Incubate membrane overnight at 4°C with agitation and anti-x primary antibody, diluted 1:2000 in freshly prepared TBS-MLK (dilution depends on protein)
# Wash membrane twice (5-10min each) with TBS-Tween
# Wash membrane twice (5-10min each) with TBS-Tween
# Incubate membrane for 1hr at room temperation with agitation in anti-goat-HRP conjugate (1:2000 dilution in TBS) (dilution depends on protein) and Bio-rad precision protein Strep Tactin-HRP (1:30,000 dilution in TBS-Tween binds to protein ladder)
# Incubate membrane for 1hr at room temperation with agitation in anti-goat-HRP conjugate secondary antibody(1:2000 dilution in TBS) (dilution depends on protein) and Bio-rad precision protein Strep Tactin-HRP (1:30,000 dilution in TBS-Tween binds to protein ladder)
# Wash membrane 3x (5-10min each) with TBS-Tween
# Wash membrane 3x (5-10min each) with TBS-Tween
'''Chemiluminescence Detection'''
'''Chemiluminescence Detection'''
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