SDS PAGE by yaofenjin

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									   Lab #6

  SDS-PAGE,
  Bradford and
  protein activity
  assays

BTC555               Lab#6
   SDS-PAGE




BTC555        Lab#6
   SDS PAGE
  • Sodium Dodecyl Sulfate Polyacrylamide
    Gel Electrophoresis is a technique that is
    used to separate and identify proteins.
  • It separates proteins according to their
    electrophoretic mobility (a function of
    length of polypeptide chain or molecular
    weight as well as higher order protein
    folding, posttranslational modifications and
    other factors).
BTC555                                         Lab#6
   Sodium dodecyl sulfate
• denatures proteins (ionic detergent, causing
  multimeric proteins to dissociate into their
  subunits).
    – All polypeptide chains are forced into extended
     conformations with similar charge : mass ratios.
     SDS treatment eliminates the effect of differences
     in shape (chain length reflects mass).
                                               -




BTC555                                              Lab#6
   SDS
• SDS ions coat the protein,
  which adopt rodlike shapes,
  so all proteins have the
  same rodlike shape.
• The negative charge of the
  bound SDS ions hides the
  variable charge of the
  polypeptide. Another words
  all polypeptides behave as
  if they had similar charge
  per unit length.
BTC555                          Lab#6
   SDS




BTC555   Lab#6
   Polyacrylamide Gels
   • Polyacrylamide chains and simultaneously
     cross-linking the chains into a semisolid matrix.
   • The pore size of a gel can be varied by adjusting
     the concentrations of polyacrylamide and the
     cross-linking reagent.




BTC555                                              Lab#6
   Acrylamide




BTC555          Lab#6
   Protein separation
                    • When a mixture of
                      proteins is applied to
                      a gel and an electric
                      current applied,
                      smaller proteins
                      migrate faster than
                      larger proteins
                      through the gel. The
                      rate of movement is
                      influenced by the
                      gel’s pore size and
                      the strength of the
                      electric field.
BTC555                                     Lab#6
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   Marker
  • In addition to the unknown protein
    samples, a set of proteins of known size
    are included in a separate lane of the gel.
    These proteins are used to create a
    calibration plot to match distance migrated
    to molecular mass.
  • Even chains that differ in molecular weight
    by less than 10 percent can be separated
    by this technique.

BTC555                                        Lab#6
   Animation




BTC555         Lab#6
   Stacking & resolving gel
• Two sequential gels are used:
    – the top gel, (stacking), 6.8 pH, has a low (5.5%)
      acrylamide concentration to make a porous gel; proteins
      separate poorly but form thin, sharply defined bands.
    – the lower gel, (separating, or resolving),8.8 pH has a
      higher polyacrylamide content (in our case, 12%), which
      causes the gel to have narrower channels or pores.
• As a protein, concentrated into sharp bands by the
  stacking gel, travels through the separating gel

                    Discontinuous system
BTC555                                                      Lab#6
   Stacking gel




BTC555            Lab#6
                   Staining
• Coomassie Brilliant Blue (CBB)
  sensitivity~100 ng protein
• 0.2% CBB in 45:45:10 %
  methanol:water:acetic acid mix
• Cover gel with staining solution, seal in plastic
  box and leave overnight on shaker (RT) or for
  2 to 3 hours at 370C also with agitation.
• Destain with 25:65:10% methanol water acetic
  acid mix, with agitation.
Coommassie brilliant blue
       G-250
   Resolving protein bands
   on SDS-PAGE gels




         M
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   Tips

  • Use water/
    butanol to
    even the
    surface of
    resolving gel.
  • insert comb
    into stacking
    gel at an
    angle to help
    avoid trapping
    air bubbles at
    end of comb
    teeth
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   Tips
• unpolymerized acrylamide is a neurotoxin - wear gloves when
  handling
   – once polymerized, gels can be disposed off in the regular trash
• keep excess solution to monitor polymerization process
• Always use non-latex gloves when handling gels or IPG strips,
  keratin and latex proteins are potential sources of
  contamination.
• add bromophenol blue to stacking solution to visualize of wells
• "smiling" and "frowning" of gels largely due to unequal heat
  distribution / salt concentration across gel
   – run gels slower or with cooling in the cold room
   – fill empty wells with extract buffer



BTC555                                                                 Lab#6
   MW determination




BTC555                Lab#6
   MW determination




BTC555                Lab#6
   • Plot log(MW) vs
     Rf (relative mobility
     for known size band

   • Interpolate unknown
     protein from its Rf
     value                   JBC,1971,Vol. 246. No 20, pp. 6328-6334,
                             Molecular Weight Determination of Protein-
                             Dodecyl Sulfate Complexes by Gel
                             Electrophoresis in a Discontinuous Buffer
                             System
BTC555                                                           Lab#6
   Acrylamide gel protein
   detection methods sensitivity
   • Coomassie Brilliant Blue (R-250) -100ng.
   • Colloidal Coomassie Brilliant Blue (G-250)
     ~30ng
   • Blue Silver (Coomassie G-250) - 1-5ng
   • Silver - sensitivity of 5-10 ng
   • Fluorescent detection methods/Ruthenium II
     - 10-20 ng


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   Outline
   •     Casting the gel
   •     Sample prep
   •     Loading the gel
   •     Running the gel
   •     Staining


BTC555                     Lab#6
   Lab # 6


 Protein
 quantification




BTC555            Lab#6
   The Bradford Assay
   • Absorbance shift in Coomassie Brilliant Blue G-
     250 (CBBG) when bound to arginine and
     aromatic residues
   • The anionic (bound form) has absorbance
     maximum at 595 nm whereas the cationic form
     (unbound form) has and absorbance maximum
     at 470 nm
   • The assay is monitored at 595 nm in a
     spectrophotometer, and thus measures the
     CBBG complex with the protein.


BTC555                                             Lab#6
   Coomassie Brilliant Blue G-
   250 (CBBG)
   • CBBG specifically binds to proteins at
     arginine, tryptophan, tyrosine, histidine and
     phenylalanine residues.
   • !! The assay primarily responds to arginine
         residues (eight times as much as the
         other). If you have an arginine rich protein,
         you may need to find a standard that is
         arginine rich as well.

BTC555                                               Lab#6
   Bradford assay
• There are two major formats of this assay:
• The micro assay format is designed for protein
  concentrations between 1-20 micrograms.
• The macro assay is designed for protein.
  concentrations in the range of 20-100 micrograms.
• It is more convenient to use the micro format and
  dilute your protein down to the concentration range.
• The assay can also be performed in a microwell plate,
  which is a very convenient way to process a large
  number of samples rapidly.
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   Micro Assay Procedure
 • Warm up the spectrophotometer for 15 min. before
   use
 • Dilute samples with buffer to an estimated
   concentration of 1 to 20 micrograms/milliliter
 • Prepare standards containing a range of 1 to 20
   micrograms protein (albumin or gamma globulin are
   recommended) to a volume of 200 µl (100µl NaOH)
 • Prepare unknowns to estimated amounts of 1 to 20
   micrograms protein per tube to 200 µl (100µl NaOH)
 • (Optional) Add 100 µl 1 M NaOH to each sample
   and vortex.
 • Add 800 µl dye reagent and incubate 5 min.
 • Measure the absorbance at 595 nm.
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   Considerations
   • Use a small amount of sodium hydroxide
     in the assay to help solubulize your
     protein. Some proteins, especially
     hydrophobic, membrane or “sticky”
     proteins tend to precipitate in the presence
     of coomassie dyes. If you observe a
     precipitate forming when you add the dye
     reagent to your sample try adding the
     specified amount of sodium hydroxide.

BTC555                                         Lab#6
   General Considerations
   • The CBBG dye used in the assay binds to
     quartz cuvettes quite strongly.
     Therefore, glass or plastic cuvettes should
     be utilized. Since this assay has a general
     tendency to bind to cuvettes, it is highly
     recommended to use disposable plastic
     cuvettes.



BTC555                                        Lab#6
   Typical Bradford Assay Standard
   Curve
                  The absorption spectra of the two
                  forms of the dye overlap. This causes
                  the assay to respond non-linearly in
                  the standard curve.

                  The assay does perform linearly over
                  short concentration stretches, and this
                  has most likely resulted in the overall
                  conclusion that the assay is linear.

                  Bradford himself even notes that the
                  assay is not linear over the whole
                  range in the original article.




BTC555                                               Lab#6
   Coomassie Blue
   • Dye stock - Coomassie Blue G (C.I.# 42655) (100 mg)
     is dissolved in 50 mL of methanol. (If turbid, the solution
     is treated with Norit (100 mg) and filtered through a
     glass-fiber filter.) The solution is added to 100 mL of
     85% H3PO4, and diluted to 200 mL with water. The
     solution should be dark red, and have a pH of -0.01. The
     final reagent concentrations are 0.5 mg/mL Coomassie
     Blue G, 25% methanol, and 42.5% H 3PO4. The solution
     is stable indefinitely in a dark bottle at 4°C.
   • Assay reagent - The assay reagent is prepared by
     diluting 1 volume of the dye stock with 4 volumes of
     distilled H2O. The solution should appear brown, and
     have a pH of 1.1. It is stable for weeks in a dark bottle at
     4°C.
   • The dye reagent is usually more convenient to purchase
     than to make, due to the use of phosphoric acid
BTC555                                                         Lab#6
                                                 Protein
   •       Red <=>      Green   <=>        Blue     <=>    Blue-Protein
         (470 nm)      (650 nm)            (590 nm)            (590 nm)
                  H+                  H+


   • Under strongly acid conditions, the dye is most
     stable as a doubly-protonated red form. Upon
     binding to protein, however, it is most stable as
     an unprotonated, blue form.




BTC555                                                                    Lab#6
   Choice of standard
   • It has been noted that Bovine Serum
     Albumin (BSA) has a double than
     “normal” response in the assay and may
     not always be suitable. Several
     researchers therefore use Imunnoglobulin
     G (IgG) as the preferred standard for the
     assay.



BTC555                                       Lab#6
   Protein Standards
   • Protein standards should be prepared in
     the same buffer as the samples to be
     assayed. A convenient standard curve
     can be made using bovine serum albumin
     (BSA) with concentrations of 0, 250, 500,
     1000, 1500, 2000 µg/mL for the standard
     assay, and 0, 10, 20, 30, 40, 50 µg/mL for
     the microassay.


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   Advantages

   •     Fast and inexpensive
   •     Highly specific for protein
   •     Very sensitive
   •     Dye reagent complex is stable for
         approximately one hour



BTC555                                       Lab#6
   Disadvantages

   • Absorbance spectra of the two Coomassie
     Brilliant Blue G-250 species partially overlap
     making the standard curve very important
   • Non-linear standard curve over wide ranges
   • Response to different proteins can vary widely,
     choice of standard is very important




BTC555                                                 Lab#6
   Lab # 6


 Protein
 Activity Assay




BTC555            Lab#6
   D-Cellobioside Enzymatic
   Assay
                     • The enzyme
                       activity can be
                       determined by
                       measuring
                       absorbance at
                       420 nm of p-
                       nitrophenol
                       (yellow color)


BTC555                                   Lab#6

								
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