Difference between revisions of "Developing Your Immunoblot Membrane"
From SDMRC
(Created page with "==Developing your Immunoblot Membrane== ===Materials=== *A flash drive *A tube rack *Sheet protectors *PICO developing solution *15 ml tube *Ethanol + KimWipes towels # Afte...") |
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===Materials=== | ===Materials=== | ||
*A flash drive | *A flash drive | ||
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*15 ml tube | *15 ml tube | ||
*Ethanol + KimWipes towels | *Ethanol + KimWipes towels | ||
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# After the 1 hour secondary time has passed take your membranes into trays of TBST to wash three times for 10 minutes | # After the 1 hour secondary time has passed take your membranes into trays of TBST to wash three times for 10 minutes | ||
# Cut new sheet protector for your membranes (they can be in one protector) | # Cut new sheet protector for your membranes (they can be in one protector) | ||
Latest revision as of 20:18, 23 July 2018
Materials
- A flash drive
- A tube rack
- Sheet protectors
- PICO developing solution
- 15 ml tube
- Ethanol + KimWipes towels
Method
- After the 1 hour secondary time has passed take your membranes into trays of TBST to wash three times for 10 minutes
- Cut new sheet protector for your membranes (they can be in one protector)
- Take your pico developing solutions (two bottles on bench: brown and clear) with a 15 ml tube, a pipette, and a tube rack to the scanner
- Quickly take your membranes into the plastic (dry a little) and bring to the scanner
- AT THE SCANNER mix the brown and white solution in the 15 ml tube and flush
- The developing solution is 1:1 so divide the total amount needed by 2
Example: 4.5 ml total so 2.25 ml of brown and 2.25 ml of clear
Developing Machine
- Open the tray and clean with ethanol
- Open the sheet protector and quickly add drops of the developing solution until is covers the membranes
- Leave the sheet open and push tray in
- On the scanner hit live view to see your membranes and adjust position if needed
- Hit application_chemiluminesence_ blot_exposure_ show preview optimal auto exposure
- Place red crop box over the area you want captured and hit camera and wait
- (optional) manual exposure_ set manual exposure time (ex. 60 sec) _ capture and wait. Make sure you are not moving the membrane during these pictures.
- Take a full picture at the end. Application_ colormetric_ exposure (automatically/ faint bands) _ capture
- Go to gallery_ take any picture and the one you just took (select both)_ hit merge_ back to gallery
- Put all pictures wanted onto the flash drive_ save/send_ save to USB stick
- Trash membranes, clean tray with ethanol
Add the ponceau scan to your other developed images
- Copy a paste ponceau scan to your flashdrive
- Make new folder, title with condition and cell type
- Put all the images from flash drive onto personal computer
