Kendrick Laboratories, Inc. is a contract research organization specializing in protein analysis using 1D and 2D SDS-PAGE.Pharma/Biotechnology,academia,food/nutritionand other.
Two-dimensional electrophoresis (2DE) is a biochemical method for separating complex mixtures of proteins into individual species.Proteins are first separated by charge using isoelectric focusing (IEF), then by size using sodium dodecyl sulfate (SDS) gel electrophoresis. The star pattern of the protein dots is visualized on the final 2D gel by staining or Western blotting. Proteins of interest can be identified by mass spectrometry.
There are currently two variants of 2DE in use around the world, differing in IEF protocol. 1) The classical IEF method,shown in the photo belowand used at Kendrick Labs, uses carrier ampholines polymerized in acrylamide tube gels to create the pH gradient. A nonionic detergent added to the mix confers compatibility with SDS, thus the method is called 2D SDS PAGE. 2) The IPG-2DE is used in most central laboratories. In this method, the IEF pH gradient is immobilized on commercially available easy-to-handle solid supports called IPG strips. Unfortunately, the strips are not SDS compatible, so sample preparation is often problematic. For more details on how these two methods compare, seeTechnical document (PDF).
2D SDS PAGE (2D Ampholine Carrier)it is compatible with SDS, by far the best reagent for protein solubilization . The method originally developed by O'Farrell  was found compatible with SDS by the Andersons  and refined at Kendrick Labs [3, 4]. Acrylamide tube gels are polymerized with carrier ampholines that form a pH gradient when a voltage is applied, and during IEF, ampholines and proteins migrate to a stationary position. The 2D SDS-PAGE method was standardized at Kendrick Labs through written SOPs. SameSpots software allows exact pattern alignment prior to matching; The computational analysis of variable complex patterns has become easy. While DIGE is possible, it is not required and the 2D SDS PAGE can be validated without it.
SDS compatibility with CA-2DE, first reported by Anderson et al. , it was further optimized at Kendrick Labs [3, 4]. Therefore, samples are homogenized in SDS buffer, heated at 100 °C until the solution is clear, and then loaded without centrifugation. The resulting 2D SDS-PAGE gels provide quantitative results.
The following pages demonstrate what the CA-2DE is:
- Compatible con SDS
- A quantitative method for analyzing most proteins.
- Extremely sensitive when used with Western Blot
2D-SDS-PAGE is compatible with SDS because this detergent, although stoichiometrically bound to proteins, is released during IEF, as shown in Figures 1-3.
|Figure 1. SDS binding mechanism. When proteins are heated in the presence of SDS and ß-mercaptoethanol (BME), the SDS binds to the peptide backbone and imparts a uniform charge-mass ratio. BME reduces disulfide bridges. All secondary and tertiary structures are lost.||Figure 2. Example: carbamylated creatine phosphokinase standard dissolved in SDS (top) or 9 M urea buffer (bottom) before IEF. The arrowhead marks the lower isoform of tropomyosin, pI markers of PM 33 kDa and pI 5.2. SDS does not interfere with the IEF of this protein.|
How can negatively charged SDS be used to resolve proteins prior to IEF when a single charge change is detected? During CA-IEF, SDS is removed from proteins to produce micelles with NP-40, a nonionic detergent . The charged micelles migrate to the acid end of the gel tube where they form a sphere that is discarded.
Figure 3. Rat liver microsomes prepared in three ways. For this figure, microsomes (pellet obtained after homogenization, low-speed rotation, and then 100,000 x g rotation) were purchased and dissolved in heated SDS buffer (left) in urea buffer (middle) or warm SDS buffer and further dilution with urea buffer (right). ). Standard shape gels (13 x 15 cm) loaded with 50 µg protein and silver stained are shown. The IEF was performed with ampholines pH 3.5-10. Microsomes made with SDS and urea show a more complex pattern than the other two. TIP: Once the sample preparation is optimized, do not change it. A change in sample preparation can change the pattern due to hidden salts and lipids affecting the pH gradient.
Examples of SDS vs. Urea
A. Purified CPK pI marker
Figure 4. pI standard of carbamylated creatine phosphokinase purchased from Amersham Biosciences and dissolved in SDS (top) or 9 M urea (bottom) prior to isoelectric focusing. The arrow marks the lower MW isoform of tropomyosin, our internal marker of MW 33,000 and pI 5.2. No differences are observed between the two buffers for this purified protein.
B. Rat liver cytosol
Figure 5. Rat liver cytosol. For this figure, rat liver cytosol (product of 100,000 x g spin) was purchased (CellzDirect/ThermoFisher) and dissolved in our standard heating SDS buffer (left) or urea buffer (right) at 1 mg/ ml. In this case, the general 2D pattern is the same, but several abundant proteins appear only in the SDS pattern, while a high molecular weight protein appears only in the urea pattern. Proteins can change their positions in SDS versus urea buffer, but samples prepared in the same way produce a reproducible pattern from run to run.
Whole cell preparation of C.E. coli
Figure 6. E. coli whole cell lysates. E. coli were pelleted, lysed, and dissolved in our standard warm SDS buffer (left) or urea sample buffer (right). Again, the patterns are similar, but some abundant proteins are unique to each pattern (arrows).
D. CHO outer membranes
Figure 7. Outer membrane samples of Chinese hamster ovary cells. Identical outer membrane pellets were resuspended in our standard SDS buffer with heat (right) or urea sample buffer (left). Similar samples dissolved in urea buffer always showed the same central bleached region on 2D gels. However, the samples made in SDS always showed a nice and reproducible pattern. These gels are courtesy of Dr. Laura Liscum, Tufts University, Boston, MA, who prepared the membrane samples.
So what is going on? The composition of the membrane influences the pattern. As shown in Table 1 (below), the outer cell membranes contain only 50% protein and also contain approximately 40% lipid and 10% carbohydrate by weight. We believe that in urea-prepared CHO membrane samples, charged lipids become concentrated during IEF and alter the pH gradient. However, in samples prepared with SDS, the loaded lipids are cleaved in the SDS/NP-40 micelles, migrate to the extreme acid end of the gel tube, and discard on the SDS bead. In addition to increasing the solubilization of membrane proteins, SDS removes interfering substances by partitioning in some samples.
|Red blood cells||49||43||8|
Table 1. Composition of the membrane. Protein, lipid and carbohydrate levels in various biological membranes (approximate percentage on dry weight). Reference "Laboratory Books Membrane Structure and Fluidity".
2D SDS-PAGE with Coomassie blue staining is quantitative for most proteins.
The relationship between the density of spots and the total protein load is excellent; R.2= 0,9971.
Figure 8. Top: montage showing the dot contours for 200 µg (top 3 panels), 400 µg (middle 3) and 600 µg (bottom 3) total protein loading for dot 8 polypeptide with MW 76,100, pI 8, 1. Bottom: Plot of dot density of integrated protein on background versus total protein loaded (200 µg, 400 µg and 600 µg) for the same dot.
- Rat liver cytosol was diluted with buffer containing 2.5% SDS + 4.5 M urea before running triplicate large format 2D gels with 200, 400 and 600 µg loadings. Kendrick Labs standard operating procedures were used for all steps. Gels were stained with Coomassie blue and scanned with a linearly calibrated laser densitometer at 0-3 OD. Sixty polypeptide spots were quantified using Nonlinear Dynamics Progenesis software.
More details on quantification can be found in our validationSalidamiPublication.
Western blotting 2D SDS PAGE has high sensitivity and specificity.
Proteins from a sample are resolved by 1D or 2D electrophoresis and then transferred to a PVDF membrane at Kendrick Labs. After transfer, the PVDF is stained with Coomassie Blue and scanned to record the overall 2D pattern. The blot is washed during subsequent incubations with blocking agent and primary antibody; does not bother Protein dissociation constants for antigen-antibody binding range from 10-8bis 10-12this extraordinarily tight binding of antibodies to an antigenic determinant (epitope) is useful for protein detection . Finally, the membrane is incubated with a secondary antibody that glows in the presence of a chemical called ECL, followed by exposure to X-ray film. (Film development is faster and more efficient than chemiluminescence scanning when the Western blots are run on large sets). Patterns on the film can be overlaid with thePhotoof the colored stain. Matching the film to the blot image and then to a duplicate stained gel for local sectioning allows for easy protein identification using mass spectrometry.
2D SDS PAGE with Western Blotting has many purposes.For example, it is useful to follow protein purification by immunoprecipitation, find proteins with post-translational modifications such as lysine acetylation, and determine if the proteins are present in a tissue at low levels. High affinity antibody binding results in high antigen sensitivity.
Western blotting of receptor tyrosine kinase:The example shown below are 2D SDS-PAGE Western blots prepared with aPhosphotirosinaAntibody against whole cell lysate from a human lung cancer tumor sample and matched control. A ~200 kDa glycosylated protein with approximately the molecular weight of a receptor tyrosine kinase (red arrow) shines brightly in the cancer sample. No signal was observed in this area for normal tissue taken from the same lung. Western blot 2D-SDS-PAGE of the cancer sample with an anti-epidermal growth factor receptor (EGFR) antibody showed a signal migrating along with the phosphotyrosine dot, suggesting that the protein is EGFR. However, three attempts to confirm identity by point-cutting and mass spectrometry (LC/MS/MS) have failed. Protein spots extracted from Kendrick Labs 2D gels can usually be identified by MS. But in this case, the phosphotyrosine western blot provides a clear signal that is much more sensitive than mass spectrometry.
Figure 9. These films obtained with a phosphotyrosine antibody demonstrate the remarkable sensitivity of 2D-SDS-PAGE Western blotting. Right: Western phosphotyrosine (pTyr) blot of a whole cell lysate from a human lung tumor sample prepared with SDS buffer. The arrow marks a putative tyrosine kinase receptor, probably the epidermal growth factor receptor, known to be a driver of lung cancer. Left: corresponding pTyr WB from matched normal lung tissue from the same patient. Conditions: standard format 2D gel (13 x 15 cm), 200 µg protein loading, pTyr PY20 antibody with overnight incubation. Binding to the strongly charged MW markers on the right allows match between batches. CalBiochem sells two of the marker proteins, the 94 kDa phosphorylase A and the 29 kDa carbonic anhydrase, as pTyr MW markers. The protein marked with the red arrow co-migrated with the protein highlighted by EGFR Western blotting in a duplicate blot (not shown).
Membraneprotein on SDS
Sample preparation is key to successful 2D electrophoresis of membrane proteins.
An effective membrane protein sample preparation consists of 2 parts: 1- preparation of the membranes and 2- dissolution in a 2D buffer.
1-Prepare the membranes:
The final purity of the membrane fraction depends on the protocol used and also on the morphology of the initial cells. The morphology can vary greatly. The book "Subcellular Fractionation" by J. Graham and D. Rickwood, Oxford University Press, 2002 edition is a good starting point for membrane preparation protocols if you do not have a specific reference for your cells or tissues.
2- Dissolve the prepared membrane proteins in SDS buffer.
The ampholine carrier IEF system used at Kendrick Labs is compatible with SDS (sodium dodecyl sulfate, CH3(CH2)11VERY3Na), and we prefer this detergent for dissolving protein samples. When proteins are heated in the presence of SDS and β-mercaptoethanol (BME), the SDS binds to the peptide backbone and imparts a uniform charge-mass ratio, as shown in the schematic below. BME reduces disulfide bridges. All secondary, tertiary and quaternary structures are lost.
For tube gels in KLabs: SDS buffer minus BME is added to the sample and then heated in a boiling water bath until the solution is clear (2-5 min). Protein determination is then performed using the BCA method, followed by the addition of BME and sometimes urea before loading on the IEF. Note that samples for IPG strips are initially diluted with urea/thiourea + CHAPS buffer and cannot be heatedted because carbamylation will occur. Carbamylation is bad - NEVER COOK SAMPLES WITH UREA.
How can negatively charged SDS be used to resolve proteins prior to isoelectric focusing, where a single charge change can be detected? Leigh and Norman Anderson  demonstrated that during gel tube IEF, SDS is removed from proteins to produce micelles containing NP-40, a nonionic detergent. The micelles are highly charged and migrate to the acid end of the tube gels where they form a sphere that is discarded.
Examples of membrane samples made with SDS
Fusobacteria outer membranes
Figure 10. Fusobacterium outer membranes. This sample is from a gram-negative anaerobic bacterium found in enteral plaque, a common pathogen of anaerobic abscesses. A type of complex glycolipid is shed along with the cell membrane and interferes with the IEF. With the permission of Dr. Susan Haake, UCLA School of Dentistry. Conditions: Standard size gel, Amfoline pH 4-8, 10% acrylamide, silver stained. On this older gel, the MW markers appear as lines across the gel, not as bands along the sides.
Red blood cell ghosts
Figure 11. Red blood cell membranes. Red blood cell ghosts are composed of 49% protein, 43% lipid, and 8% carbohydrate. Note that many membrane proteins are glycosylated and are not discrete blots due to microheterogeneity in the sugar groups. Duplicate gels are shown to show reproducibility; Arrows mark reproducible striatal proteins. Courtesy of Dr. Asok Chaudhuri, New York Blood Center, Department of Cell Biology. Conditions: Standard size gel, pH 3.5-10 Ampholine, 10% acrylamide, silver stained.
Mouse brain synaptosomes
Figure 12. Mouse brain synaptosomes (nerve ending particles). Two different preparations with two different percentages of acrylamide are shown for the second dimension gel, 11% (left) for resolving intermediate molecular weight proteins and 7% (right) for resolving high molecular weight species. Other interfering substances are present in the sample on the right. These gels are made with the permission of Dr. Christopher Ellis, National Institutes of Health. Conditions: Standard size gel, pH 3.5-10 Ampholine, 10% acrylamide, silver stained.
inner membranes of hepatocytes
Figure 13. Internal membranes of human hepatocytes. The membrane fraction was obtained by homogenization, differential centrifugation and a final step of sucrose density gradient fractionation. The final fraction was dialyzed against 20 mM Tris-HCl pH 8, 50 mM NaCl, 5 mM EDTA, and 5% glycerol prior to shipment. At Kendrick Labs, the sample was redissolved to 1 mg/mL in SDS buffer and heated in a boiling water bath for 5 minutes before loading 50 µg. Conditions: Standard size gel, pH 3.5-10 Ampholine, 10% acrylamide, silver stained.
Examples SDS + urea
Example of a membrane sample prepared with SDS followed by the addition of 4.5 M urea.
mouse liver microsomes
Figure 14. Rat liver microsomes prepared in 3 ways. For this figure, rat liver microsomes (pellet obtained after homogenization, low-speed rotation, and post-rotation at 100,000 x g) were purchased (CellzDirect/ThermoFisher) and dissolved in the standard at 1 mg/mL in one of our standard SDS buffers with heating (left) Urea buffer (middle) or SDS buffer with heating followed by urea buffer (right). Standard size gels, pH 3.5-10 Ampholine, 10% acrylamide, silver stained.
The final 2D gel of microsomes made with SDS and urea is significantly better than gels from either buffer alone.
1. O'Farrell, P.H., Two-dimensional electrophoresis of high-resolution proteins. J. Biol. Chem., 1975. 250: p. 4007-2
2. Anderson, L. and N. G. Anderson, High-resolution two-dimensional electrophoresis of human plasma proteins. Proc Natl Acad Sci USA, 1977. 74(12): p. 5421-5.
3. Burgess-Cassler, A., J.J. Johansen, DA Santek, JR Ide, and NC Kendrick, Computerized quantitative analysis of Coomassie blue-stained serum proteins separated by two-dimensional electrophoresis. Clinical Chem., 1989. 35(12): p. 2297-304.
4. Kendrick, N., CC Darie, M Hoelter, G Powers, and J Johansen. 2D SDS PAGE in combination with Western Blotting and Mass Spectrometry is a robust method for protein analysis with many applications. Advances in experimental medicine and biology. 2019. 1140: pp. 563-574.
5. Lehninger, A.L., D.L. Nelson and M. M. Cox, Lehninger's Principles of Biochemistry. 5th ed. 2008, New York: W. H. Baron.
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