Seminar 10:
Analysis of proteins – extraction, electrophoresis and basic analysis methods
Depending on the aim of study, properties of protein under investigation and detection method one has to
decide whether native, fixed or denatured protein will be analysed. Proteins can be extracted from cells
(adherent or suspension cultures) or tissues. For this reason different buffers and procedures will be used in
each case.
This workshop will focus on procedures concerned with electrophoretic analysis of proteins. Detailed
protocols are outlined below.
1a. Preparation of lysate from cell culture
1. Place the cell culture dish in ice and wash the cells with ice-cold PBS.
2. Drain the PBS, then add ice-cold lysis buffer (1 ml per 107 cells/100mm dish/150cm2 flask; 0.5ml
per 5x106 cells/60mm dish/75cm2 flask).
3. Scrape adherent cells off the dish using a cold plastic cell scraper (or rubber policeman), then gently
transfer the cell suspension into a pre-cooled microfuge tube.
4. Maintain constant agitation for 30 minutes at 4°C.
5. Centrifuge in a microcentrifuge at 4°C.
You may have to vary the centrifugation force and time depending on the cell type; a guideline is
20 minutes at 12,000 rpm but this must be determined by the end-user (e.g. leukocytes need a
very light centrifugation).
6. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in
a fresh tube kept on ice, and discard the pellet.
The buffer (with inhibitors) should be ice-cold prior to homogenization. Lysis buffers can be denaturing (e.g. RIPA buffer
- Radio Immuno Precipitation Assay buffer) or non-denaturing (potassium phosphate buffer). Choice of proper lysis
buffer must be determined by further protein detection technique. In either case following homogenization proteins
can be extracted from cells or tissues by 3 freeze-thaw cycles. For this purpose samples are frozen by immersion in
ethanol/dry ice bath or liquid nitrogen.
2. Determination of protein concentration
Perform a Bradford assay, a Lowry assay or a BCA assay. Bovine serum albumin (BSA) is a frequentlyused protein standard.
=> See Bradford assay manual for details. Pay attention to sections marked yellow.
3. Electrophoresis
There are two types of electrophoreses: one dimensional (i.e., one plane of separation) and two
dimensional. One dimensional electrophoresis is used for most routine protein separations. It separates
proteins based on their molecular weights. Two dimensional electrophoresis separates proteins
according to isoelectric point and molecular weight.
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METHODS IN MOLECULAR BIOTECHNOLOGY
(A) 1-D electrophoresis; (B) 2-D electrophoresis – following separation in one
direction (A) proteins are separated in perpendicular plane (B), simplified schematic.
2D gel electrophoresis (2DE) is a key technique for purifying individual proteins from
complex samples based on their isoelectric points and molecular weights.
SDS-PAGE (for Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis)
The technique is a standard means for separating proteins according to their molecular weight.
Polyacrylamide gels are formed from the polymerization of two compounds, acrylamide and N,Nmethylenebisacrylamide (Bis-acrylamide, for short). Bis-acrylamide is a cross-linking agent for the gels. The
polymerization is initiated by the addition of ammonium persulfate (APS for short) along with TEMED.
The separation of molecules within a gel is determined by the relative size of the pores formed within the gel.
The pore size of a gel is determined by two factors: the total amount of acrylamide present (designated as
%T) and the amount of cross-linker (%C). As the total amount of acrylamide increases, the pore size decreases.
(A)
(B)
(A) schematic of electrophoretic protein separation in a polyacrylamide gel. MW,
molecular weight. (B) effect of SDS on the conformation and charge of a protein. SDS
binds at a constant rate of 1.4 g of SDS per 1 g protein (a stoichiometry of about one SDS
molecule per two amino acids)
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METHODS IN MOLECULAR BIOTECHNOLOGY
3a. Discontinuous denaturing gel preparation (SDS-PAGE)
1. Assemble the glass plates according to the manufacturer’s instruction (see Bio-Rad manual
for Mini-Protean system – it will be explained in short during the course).
2. Prepare the appropriate volume of resolving gel solution:
40% PAA (29:1) (polyacrylamide)
1,25 ml (10% final conc.)
1M Tris-HCl, pH 8.8
1,875 ml
10% SDS (sodium dodecyl sulphate)
50 µl
10% APS (ammonium persulfate)
50 µl
ddH2O
make up to 5 ml
add 3.3 µl TEMED, mix gently and proceed immediately to step 3
3. Pour the acrylamide solution into the gap between the glass plates. Leave sufficient space
for the stacking gel (1/4 of the gel length).
4. Carefully overlay with isobutanol .
5. After polymerization is complete (30 min.), pour off the overlay and wash top of the gel
with ddH2O. Drain the fluid from top of the gel, and then remove any remaining water with
paper towel.
6. Prepare the appropriate volume of stacking gel solution:
40% PAA (29:1)
0,625 ml (5% final conc.)
1M Tris-HCl, pH 6.8
0,625 ml
10% SDS
50 µl
10% APS (ammonium persulfate)
50 µl
ddH2O
make up to 5 ml
add 3.3 µl TEMED, mix gently and proceed immediately to step 7
7. Pour the stacking gel directly on the surface of the polymerized resolving gel up to the edge
of the shorter plate. Immediately insert teflon comb. Be careful to avoid trapping air
bubbles.
8. Let the gel polymerize for 30 min.
9. Mix samples with the appropriate volume of the SDS gel-loading buffer (6x conc.) to obtain
1x concentration.
10. Heat samples to 100° C for 3 minutes to denature proteins.
11. Prepare sufficient volume of gel running buffer by diluting 5x conc. stock to 1x
concentration.
12. After polymerization is complete, remove the teflon comb carefully.
13. Mount the gel in the electrophoresis apparatus.
14. Add running buffer to the inner and outer chamber.
15. Wash wells with running buffer by pipetting up and down.
16. Load samples.
17. Apply a voltage of 8-15 V/cm and run the gel until bromophenol blue reaches the bottom of
the resolving gel.
18. After turning off the power supply remove the glass plates from the electrophoresis
apparatus and place them on paper towel. Use an extra spacer or plastic tip to pry the
plates apart.
19. At this stage gel can be fixed, stained or used to establish an immunoblot (Western
hybridization).
http://www.youtube.com/watch?v=IWZN_G_pC8U SDS-PAGE background theory
http://www.youtube.com/watch?v=XUjLO-ek2C8 SDS-PAGE practical guide
Zakład Biotechnologii i Inżynierii Genetycznej
METHODS IN MOLECULAR BIOTECHNOLOGY