Protein purification

这是一篇关于Protein purification的思维导图，主要内容有The protein we are seperating、1.lon exchange chromatography、2.Gel filtration chromatography、3.Bradford protein concentration method等。

Protein purification

• The protein we are seperating
  • cytochrome C, myoglobin and ferritin
    • all same reddish colour
  • Myoglobin
    • single polypeptide 16900
• 1. Ion exchange chromatography
  • principle: seperates proteins by charges
  • Colomn
    • Matrix
      • diethyl amino ethyl- (DEAE) on matrix is positively charged
        • so exchange anions
          • negatively charged would be drag down
    • 100mM Tris-HCl: PH-9.0
  • Elusion
    • Elusion buffer: Nacl
    • best be increasingly high concentration
      • Salt gradience
  • Result
    • Flew through fraction
      • Cytochrome C
    • Elute fraction
      • Myoglobin
      • Ferritin
  • details
    • The column is equilibrated in 100 mM Tris-HCl, pH 9.0 and the pump is set at 1.0 mL min-1. Run the column as follows, but retain 50-100 μL of the original sample for analysis.
• 2. Gel filtration chromatography
  • Principle: seperate proteins by sizes
    • limitation: low resolusion due to diffusion
    • benifit: get nature protein
  • Pore size
    • Sephacryl, S200HR, will separate proteins in the range 5 - 250 kDa
    • Any protein larger than 250kDa will elute first (void volume)
  • So the big ferritin will first elute
  • Result
    • Peak 1
      • Ferritin
    • Peak 2
      • Myoglobin
• 3. Bradford protein concentration method
  • aim
    • assess the final purity and yield after seperation
  • procedure
    • identify 2 peaks first using the scanned absorbance
      • Scanning spectrophotometry
        • 350nm-650nm
          • results in 426nm
    • measure the dye's absorbance at 595nm which accounts for the real protein concentration
  • Standard protein solusion
    • calibration curve
      • IgG solusion in 100mA Tris-HCl
        • 280nm ab
        • The absorbance of a 1.0 mg mL-1 solution of IgG is 1.44 at 280 nm.
  • Bradford dye
    • 1.0 mL of Bradford dye reagent is added to 20 μL of a protein sample, mixed and incubated at room temperature for at least 5 min
      • Color is from brown to red
        • ab of the dye is 595nm
          • actually the ab is scanned to find the peak in our proteins cases
          • blank produced by adding 1.0 mL of dye reagent to 20 μL of the appropriate buffer
  • Result
    • OR
      • 0.55g/ml
    • FT
      • 0.17g/ml
    • Elute
      • 0.50g/ml
    • peak 1
      • 0.085g/ml
    • Peak 2
      • 0.046g/ml
• 4. SDSpages
  • Whole name
    • polyacrylamide gel electrophoresis
      • benifit: high resolusion(no diffusion)
      • limitation: dinatured and seperated subunits
  • polyarylamide gel
    • 12% polyacrylamide
      • toxic
      • but it's more dense and able to seperate small molecular weight's proteins
      • Agrose gel can only seperates molecules with large MW
        • like DNA, RNA
          • otherwise for protein will diffuse
  • Principle: seperate proteins by MW
  • How to make the gel
    • Two kinds of gel with slightly different density therefore the proteins can condense at the interface
      • Both the gel, the loading dye buffer, and the buffer around the gel contains SDS
    • After mixed the sample with the loading die buffer
      • It heats in 100 degree for 3min to completely denature the protein.
        • And let the SDS attach around the linearized protein.
  • loaded samples
    • molecular weight marker
    • original mixture of the 3
    • IEC Elutted myoglobin+ferritin
    • IEC FT cytochrome c
    • GFC peak 1 ferritin
    • GFC peak 2 myoglobin
  • Commassie blue
    • To visualize the protein bands in our sample
• 5. Western blot
  • antibodies specific for each bar of SDS
  • Key words
    • Membrane
      • Proteins in SDS polyarylamide gel is transfered by layer of fliters and sponges under electric field
        • Subtopic
    • Blocker proteins
      • BSA or Milk
      • block the membrane part which not binds to the protein
        • reduce unspecific antibodies binding to membrane
    • Primary antibody
      • Recognize cytochrome c
    • Secondary antibody
      • recognize Fc region of the primary antibody
      • has HRP in its Fc region
        • an enzyme can oxidize a substrate and release light and the bands can be visualized under UV
  • Process of immune blocking
    • All these are done in a small box
    • first add blocker protein
    • wash
      • Don't wash too much, but gently wash out the blockers covered our target proteins
        • TTBS wash buffer
    • then add primary anitibody
    • Wash 3-5times, each time shake 5 min
      • wash to get rid of unspecific bindings
    • then add secondary antibody
    • Wash
• 6. Sequencing
  • +online database