Western Blot

From VLP

Lysing Cells

 * Chill PBS on ice
 * Prepare labeled microtubes for each sample
 * Thaw enough urea lysis buffer to each sample (depending on amt of cells)
 * Get cell scraper (next to George’s bench) and paper towel sprayed with 70% EtOH
 * Aspirate media from cells
 * Rinse 2 X 1ml PBS
 * Add 1ml PBS and use scraper to get all cells off the dish
 * Transfer cells from dish to microtube and place on ice
 * Pulse-spin to pellet cells and place on ice
 * Resuspend in appropriate volume of lysis buffer by vortexing (~40-600µl depending on amount of cells)
 * Incubate on ice for 30 min.
 * Spin down to pellet cell debris
 * Transfer cell lysate to new microtubes

Measuring Protein Concentration (Bradford Assay)

 * Dilute BSA to generate standard curve
 * BSA (2mg/ml) located in 4ºC
 * Prepare Bradford Reagent (BR; dilute 1:5 in distilled water)
 * Dilute 1ul BSA in 1ml Bradford Reagent (BR) to make serial dilution
 * Further dilute 50ul in 1ml BR for 0.1ug 100ul in 1ml BR for 0.2ug 250ul in 1ml BR for 0.5ug 500ul in 1ml BR for 1ug
 * Add undiluted BSA to BR: 1ul in 1ml for 2ug 2ul in 1ml for 4ug 4ul in 1ml for 8ug 8ul in 1ml for 16ug
 * Prepare microtubes with 1ml BR for each sample
 * Add 2ul sample to each microtube with BR Incubate at rt for 5-15min
 * Measure A595 for standard curve and samples
 * Cuvettes are above Katie’s bench
 * Make sure the spectrophotometer has the right cuvette holder
 * Set the fixed wavelength to 595
 * Measure the absorbance for each sample
 * Determine protein concentration in each sample
 * Normalize to lowest amount protein concentration
 * Divide smallest absorbance by all to get ratio of protein
 * Set standard amount for normalizer(i.e. use 60µl of lowest sample and adjust other sample by multiplying the ratio of lowestsample/othersamples x 60µl; add the resulting amount of lysate to a tube and dilute to 60µl with buffer)
 * Add Loading Dye (4X LDS from Invitrogen)

Loading/Running PAGE

 * Choose appropriate acrylamide percentage based on protein size (or use gradient gel)


 * 10-well gels hold 25ul 12-well gels hold 20ul 15-well gels hold 15ul


 * For Bis-Tris gels (NuPAGE-Invitrogen, in 4C walk-in) use MOPS Running Buffer (next to Vanessa’s bench)


 * Remove white sticker from back of gel and set up gel apparatus


 * If only running one gel, use buffer dam


 * Add running buffer to tank (500ml per tank)


 * Rinse out wells by aspiration and refill with buffer


 * Add appropriate amount of sample to each well


 * Connect to power source and run at 140V for 1.5-2h; until blue dye runs out

Semi-dry Transfer

 * Prepare transfer buffer and cut Whatmann paper (blotting paper) and membrane
 * Rinse PVDF membrane in MeOH (do not do this with nitrocellulose membrane)
 * Place blotting paper and membrane in transfer buffer
 * Make paper(x2):gel:membrane:paper(x2) sandwich and squeeze out bubbles
 * Place on semi-dry transfer machine and squeeze out bubbles again
 * Place lid on top of machine and run at 10-12V for 1.5-2hrs
 * Check that current is decreasing
 * Once done, put in 5% milk in TBST

Western Blot

 * Blocking: Place membrane in 5% milk in TBST (5g milk in 95ml TBST; can be used for 1-2weeks after made) for one hour
 * Wash in TBST 3X 10 min
 * Put membrane in primary antibody diluted in TBST for one hour-o/n
 * Wash in TBST 3X 10 min
 * Put membrane in secondary antibody diluted in blocking solution for one hour
 * Wash in TBST 3X 10 min
 * Add ECL solutions A and B—shake vigorously for one min
 * Place on saran wrap and develop in dark room