Difference between revisions of "Preparation of Protein Lysates from Cells"

From Bridges Lab Protocols
Jump to: navigation, search
(Created page with "==Materials== *RIPA Buffer (see RIPA) or other Lysis buffer. Add protease inhibitors. *Cells (fresh or frozen) ==Protocol== #Cool centrifuge to 4C #If cells...")
 
 
(One intermediate revision by the same user not shown)
Line 9: Line 9:
 
#Incubate on ice for 15 minutes
 
#Incubate on ice for 15 minutes
 
#Centrifuge at 14 000 RPM at 4C for 10 min
 
#Centrifuge at 14 000 RPM at 4C for 10 min
−
#Remove supernatant to clean tube.  If lysing fat, try to avoid the floating fat cake.  If necessary respin to clarify
+
#Remove supernatant to clean tube.  If lysing fat cells, try to avoid the floating fat cake.  If necessary respin to clarify
−
#Follow Protein Lysate instructions for Bradford Assay (see [[Bradford Assay]])
+
#Prepare samples for gels by adding 160ul lysate, 40ul of 10x reducing agent and 200ul of 2x SDS sample buffer for 400ul total volume.   
−
#Prepare samples for gels by adding 800 ug protein to a final volume of 200 uL of lysis buffer.  Add 200 uL of 2X loading buffer with B-ME to each lyaste.  This will generate a 2 mg/mL protein solution in SDS Loading Buffer
+
#Heat samples with loading buffer at 85C for 2-3 mins
−
#Heat samples with loading buffer at 95C for 5 mins
+
 
#Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80
 
#Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80
  

Latest revision as of 21:43, 5 January 2018

Materials

  • RIPA Buffer (see RIPA) or other Lysis buffer. Add protease inhibitors.
  • Cells (fresh or frozen)

Protocol

  1. Cool centrifuge to 4C
  2. If cells are not already frozen in buffer, add ~400ul RIPA plus PI while keeping cells on ice
  3. Scrape cells and transfer to cold micro centrifuge tube on ice
  4. Incubate on ice for 15 minutes
  5. Centrifuge at 14 000 RPM at 4C for 10 min
  6. Remove supernatant to clean tube. If lysing fat cells, try to avoid the floating fat cake. If necessary respin to clarify
  7. Prepare samples for gels by adding 160ul lysate, 40ul of 10x reducing agent and 200ul of 2x SDS sample buffer for 400ul total volume.
  8. Heat samples with loading buffer at 85C for 2-3 mins
  9. Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80
Retrieved from "http://bridgeslab.sph.umich.edu/protocols/index.php?title=Preparation_of_Protein_Lysates_from_Cells&oldid=1409"