Spotlight Biotechnology & Microtechnology
Two-dimensional electrophoresis is a critical technique for proteomic research; many researchers believe two-dimensional (2D) electrophoresis can only be accomplished using a combination of 2D gel electrophoresis to separate proteins, followed by visualisation through either (a) mass spectrometry (MS) or (b) the use of a cooled CCD imaging system combined with image analysis software for protein identification. Although these techniques are powerful and sensitive, proteins can be identified easily and quickly by staining a 2D gel with either Coomassie blue or silver stain. 2D gels were used to determine a quick method to differentiate human alpha-NAGAL protein from IGG antibodies (with similar molecular weights) contaminates obtained during fractionation.
A Quick and Easy Method to Separate Proteins of Similar Molecular Weights Using 2D Electrophoresis
MATERIALS AND METHODS
IPG BlueStrips were from Serva Electrophoresis GmbH (Heidelberg, Germany). CodeBlue stain was from Pierce (Rockford, IL). All other reagents used were from Hoefer, Inc (Holliston, MA).
Preparation
Samples of Human alpha-NAGAL protein, IGG antibodies, and a mixture of both were loaded onto 7cm (3-10NL) immobilized pH gradient (IPG) strips using overnight rehydration. Three samples were prepared; (a)10µg of pure alpha-NAGAL protein (b)10µg of rabbit IGG (c)5µg of pure alpha-NAGAL protein and 5µg of rabbit IGG. Each sample was mixed in 130µls of fresh rehydration buffer (Table 1) and then 130µl of rehydration buffer was added to each channel of the rehydration tray (Table 2).
Each sample was placed on different IPG strips, the IPG strips were placed gel side down in the rehydration buffer and then overlaid with mineral oil to prevent evaporation during the overnight rehydration at room temperature.
Note - It is important not to place the IPG strips into a refrigerator or the urea will crystallise.
First Dimension Electrophoresis - Running the IPG Strips in the Isoelectric Focusing Unit
“An impressive feature of the IEF100 unit is its unique ability to monitor each IPG strip individually in real time and maintain a record of each strip’s focusing data for later review and publishing.”
The 6 rehydrated 7cm IPG strips were removed from the rehydration tray and inserted gel side up into the channels of the focusing tray with the anodic (+) end to the left side of the Hoefer IEF100 Isoelectric focusing unit.
A filter paper wick was placed on both the anodic and cathodic end of each IPG strips. Each wick was moistened with distilled de-ionised water and lightly blot dry with a paper towel. The electrodes were placed onto the filter paper wicks and secured by clipping into place on the focusing tray. After making sure the electrodes were secured, each focusing tray channel was filled with 10mls of mineral oil.
To separate the proteins on the IPG strips, pre-programmed protocol 2 was used: 7cm Constant Wattage (Table 3). During the run, four 10% 8 X 10cm Tris-Glycine SDS-PAGE gels were cast with the Hoefer SE245 Dual Gel Casters.
A 1cm space was left at the top of each gel to accommodate the IPG strip. At the end of the run, the electrodes were removed and, using forceps, the strips were placed into a clean rehydration tray and equilibrated for 15 minutes in EBI (Table 4), then 15 minutes in EBII (Table 5). At the end of the equilibration steps, the three strips were stained with CodeBlue stain to observe the banding (Figure 1).
Second Dimension Electrophoresis - Running the SDS-PAGE Gel
One IPG strip was placed onto a 8 x 10cm Tris-Glycine SDS- PAGE gel using forceps. The protein marker was placed in a 90°C water bath for 3 minutes before applying 200ng to a 5mm x 5mm piece of filter paper. The IPG strip and filter paper with marker were held in place with a molten 0.5% agarose solution overlay ensuring contact between the IPG strip and the gel.
The Hoefer SE250 Vertical electrophoresis unit was run at 200 volts for 40 minutes. After the run, the gel was removed and stained with CodeBlue (Figure 3).
1D separation – Running the SDS-PAGE gel
A 10% PAGE resolving gel with 4% stacking gel was cast with the Hoefer SE245 Dual Gel Caster and then placed into a Hoefer SE250 mini vertical electrophoresis unit. Protein markers were placed in a 90°C water bath for 3 minutes, after which 200 ng was added to the first well of the 8 x 10 cm Tris-Glycine SDS-PAGE gel.
10µg of purified Human a-NAGAL was placed in the second well. The mini vertical electrophoresis unit was run at 200 volts for 40 minutes. After the run, the gel was removed and stained with CodeBlue (Figure 2).
Figure 2. Shows a 1D lane of purified human alpha- NAGAL protein.
alpha-NAGAL protein rabbit IGG
Author Details: Nat Clark,
Department of Biochemistry, University of Massachusetts, Amherst, Massachusetts, USA
alpha-NAGAL protein
rabbit IGG
Figure 1. IPG Strips 3-10NL stained with CodeBlue; note the banding on the strips. Top strip is pure alpha-NAGAL protein; middle strip is rabbit IGG; bottom strip is a mixture of rabbit IGG and pure alpha-NAGAL protein.
Figure 3. is the 2D gel of the purified human alpha-NAGAL protein. Note the two bands are revealed by the 2D experiment, which are not apparent in the 1D gel (Figure 2).
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68
