Difference between revisions of "Ponceau staining of protein blots"

Latest revision as of 21:12, 18 July 2018

Ponceau Staining of Protein Blots

Summary

In order to ensure the transfer went through you will stain your membrane in ponceau solution.

Materials

• Pre-made ponceau stain (50ml tube)
• Clean trays
• Forceps
• Sheet protectors
• Scanner
• TBS + tween
• TBST + BSA

1. Take plastic rectangle trays by sink, rinse, and pour enough ponceau in to cover your membrane in. Grab multiple trays per membrane you have. Ponceau can be found on the bench in a 50 ml falcon tube
2. Open the sandwich holder and carefully remove the membrane by forceps. Take it straight into the ponceau and cover (you should see proteins appearing) DO NOT LET THE MEMBRANE DRY. Place on the rocker for two minutes.
3. Re-store old transfer buffer into its bottle and clean the transfer tank/ materials
4. Prepare a sheet protector about the size of your membrane(s)
5. After the two minutes pour the ponceau back into the falcon tube and store on bench
6. Add a little DI water to your tray with the membrane (don’t spray water directly on the membrane) and swirl it around to clean
7. Pour out the DI carefully and quickly place the membrane(s) in the sheet protector. Do not let the membrane dry.
8. Take a scan of your ponceau stain
9. Turn on the computer and open the HP scanner program

Select picture to file and put the membrane on the scanner face down (make sure to clean any random liquid)

• After taking a quick scan save the scan in the folder as follows for this example
• Browse_Jordan Ponceau_file name: ponceau, C2C12, Agrin stimulation, date (on blot run)
• Scan new picture
• crop

(if there is a mark or random blotch on the scan clean and run again. Ponceau ensures we loaded the right amount of protein)

1. After taking a scan clean ponceau trays with DI
2. Add TBS tween in trays [recipe: 100 ml 10x TBS + 900 ml DI = 1x solution]
3. Carefully remove the membrane from the sheet protector and quickly mark the ladder in pencil and cut any excess membrane. (Cut membrane based on ladder if you need specific bands)
4. Place in TBS tween and set on rocker for two minutes. This removes ponceau.
5. Prepare 2.5% BSA blocking solution in a 50 ml tube. [BSA: 5 ml BSA (use a spoon found by the sink to measure the powder) + 45 ml of TBS tween. Vortex thoroughly until dissolved)
6. After the two minutes have passed pour out the TBS and place in TBST+BSA and place on the rocker for an hour