• 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
Sodium dodecyl sulfate
• denatures proteins (ionic detergent, causing
multimeric proteins to dissociate into their
– 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).
• 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.
• 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
• When a mixture of
proteins is applied to
a gel and an electric
migrate faster than
through the gel. The
rate of movement is
influenced by the
gel’s pore size and
the strength of the
• 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.
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
• 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
Resolving protein bands
on SDS-PAGE gels
• Use water/
• insert comb
gel at an
angle to help
air bubbles at
end of comb
• unpolymerized acrylamide is a neurotoxin - wear gloves when
– 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
• 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
• 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
Acrylamide gel protein
detection methods sensitivity
• Coomassie Brilliant Blue (R-250) -100ng.
• Colloidal Coomassie Brilliant Blue (G-250)
• Blue Silver (Coomassie G-250) - 1-5ng
• Silver - sensitivity of 5-10 ng
• Fluorescent detection methods/Ruthenium II
- 10-20 ng
• Casting the gel
• Sample prep
• Loading the gel
• Running the gel
Lab # 6
The Bradford Assay
• Absorbance shift in Coomassie Brilliant Blue G-
250 (CBBG) when bound to arginine and
• 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.
Coomassie Brilliant Blue G-
• CBBG specifically binds to proteins at
arginine, tryptophan, tyrosine, histidine and
• !! 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.
• 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.
Micro Assay Procedure
• Warm up the spectrophotometer for 15 min. before
• 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
• Add 800 µl dye reagent and incubate 5 min.
• Measure the absorbance at 595 nm.
• 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.
• 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
Typical Bradford Assay Standard
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.
• 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
• The dye reagent is usually more convenient to purchase
than to make, due to the use of phosphoric acid
• Red <=> Green <=> Blue <=> Blue-Protein
(470 nm) (650 nm) (590 nm) (590 nm)
• 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.
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
• 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
• Fast and inexpensive
• Highly specific for protein
• Very sensitive
• Dye reagent complex is stable for
approximately one hour
• 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
Lab # 6
• The enzyme
activity can be
420 nm of p-