Difference between revisions of "Western Blotting"
From Bridges Lab Protocols
(Corrections to protocol) |
|||
| (2 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
==SOP== | ==SOP== | ||
| − | *Irritant | + | *Irritant |
| + | *Electrophoresis | ||
==Materials== | ==Materials== | ||
| Line 12: | Line 13: | ||
##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back | ##Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back | ||
##Boil sample at 85 degrees for ~3 min | ##Boil sample at 85 degrees for ~3 min | ||
| − | ##Load 3 microliters of protein ladder (purple) (in the 4 degree), and 10 microliters of each sample into separate wells. | + | ##Load 3 microliters of protein ladder (purple top) (in the 4 degree), and 10 microliters of each sample into separate wells. |
| − | ## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel | + | ## Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel. (Tip: Gel runs more evenly if you start a lower V and increase once the samples have run down 1/3 of the gel.) |
#Make sandwich (black side, sponge, filter paper, gel, nitrocellulose, filter paper, sponge, clear side), ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer. | #Make sandwich (black side, sponge, filter paper, gel, nitrocellulose, filter paper, sponge, clear side), ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer. | ||
| − | #Transfer 4h at 75V (in cold room) or overnight at 35V (room temp). | + | #Transfer 4h at 75V (in cold room) or overnight at 35V (room temp with an ice pack). |
#Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished pour total protein stain back in bottle for later use! | #Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished pour total protein stain back in bottle for later use! | ||
#Rinse twice in revert wash solution (60ml MeOH, 13.4 ml Acetic Acid, 126.6 ml Water) | #Rinse twice in revert wash solution (60ml MeOH, 13.4 ml Acetic Acid, 126.6 ml Water) | ||
| Line 25: | Line 26: | ||
#Incubate with appropriate secondary antibody (10 000X) for 45min-1h (20 ml 2% BSA 1ul of both secondary antibodies, all found in fridge Ab stocks with blue dots on top) | #Incubate with appropriate secondary antibody (10 000X) for 45min-1h (20 ml 2% BSA 1ul of both secondary antibodies, all found in fridge Ab stocks with blue dots on top) | ||
#Wash blot every 5 minutes for 15 min with TBST. | #Wash blot every 5 minutes for 15 min with TBST. | ||
| − | #Rinse once or twice with double distilled water | + | #Rinse once or twice with double distilled water. |
| − | + | #Scan dry blot using the LiCor Odyssey [[Scanning and Analyzing Western Blots Using LiCor Odyssey]]. | |
| − | # | + | |
| − | + | ||
==If Using LiCor== | ==If Using LiCor== | ||
| − | |||
| + | [[Scanning and Analyzing Western Blots Using LiCor Odyssey]] | ||
[[ Category: Western Blotting ]] | [[ Category: Western Blotting ]] | ||
Latest revision as of 15:58, 26 March 2021
Contents
SOP
- Irritant
- Electrophoresis
Materials
- Transfer Buffer (200 mL Methanol, 100 mL 10X Transfer Buffer to final 1L volume)
- Transfer Apparatus, either Bio-Rad or Invitrogen
Protocol
- Turn on heat block to 85 degrees
- Run SDS-PAGE gel using SDS-PAGE Running Buffer and prepare diluted transfer buffer
- Use a prepared 4-12% tris gel (in the 4 degree). Remove it from the packaging, remove the white strip of tape from the bottom back, and gently pull the comb out and rinse with water.
- Load into gel tank. Fill with SDS page Running buffer (1X) to the fill line in the front and halfway up the back
- Boil sample at 85 degrees for ~3 min
- Load 3 microliters of protein ladder (purple top) (in the 4 degree), and 10 microliters of each sample into separate wells.
- Place top on tank, plug into power source and run at 125 Volts until samples and ladder reach the bottom of the gel. (Tip: Gel runs more evenly if you start a lower V and increase once the samples have run down 1/3 of the gel.)
- Make sandwich (black side, sponge, filter paper, gel, nitrocellulose, filter paper, sponge, clear side), ensuring no bubbles between layers with black piece on bottom and layer as above. Place in apparatus so that the black sandwich touches the black transfer piece. Fill with transfer buffer.
- Transfer 4h at 75V (in cold room) or overnight at 35V (room temp with an ice pack).
- Stain for total protein with Revert total protein stain on rocker for 5 minutes --when finished pour total protein stain back in bottle for later use!
- Rinse twice in revert wash solution (60ml MeOH, 13.4 ml Acetic Acid, 126.6 ml Water)
- Scan using licor for total protein, which will be used to normalize the blot
- Rinse nitrocellulose in revert reversal solution for at least 5 and no more than 10 minutes until nitrocellulose appears clear again (.2g NaOH, 60ml MeOH, 140ml Water)
- Rinse nitrocellulose in 2% BSA (2g BSA in 100ml TBST, stored in fridge) for 1 hour
- Incubate with primary antibody (check for dilution) in 2% BSA for >1h
- Wash blot every 5 minutes for 15 min with TBST.
- Incubate with appropriate secondary antibody (10 000X) for 45min-1h (20 ml 2% BSA 1ul of both secondary antibodies, all found in fridge Ab stocks with blue dots on top)
- Wash blot every 5 minutes for 15 min with TBST.
- Rinse once or twice with double distilled water.
- Scan dry blot using the LiCor Odyssey Scanning and Analyzing Western Blots Using LiCor Odyssey.
