Chapter 6. Simultaneous Saccharification and Fermentation in shake flask at higher concentration of pretreated wild grass and
1. Quest for alternative energy
2.2 Materials and Methods
2.2.5 SDS-PAGE analysis of recombinant proteins
The recombinant proteins were separated on Sodium dodecyl sulphate- Polyacrylamide gel electrophoresis SDS-PAGE gel on the basis of their respective molecular size. PAGE is an analytical method used to separate components of a protein mixture based on their size (Laemmli, 1970; Sambrook et al., 1989). The PAGE makes use of the fact that a charged molecule migrates in an electric field in the direction of an electrode with opposite charge. But this method cannot be used to determine the molecular weight of proteins because the mobility of a substance in the gel depends on both charge and size. Therefore, the proteins were treated with SDS so that they acquire uniform charge, then the electrophoretic mobility depends primarily on size. The proteins being covered by SDS are negatively charged and when loaded onto a gel and placed in an electric field, they migrate towards the anode and are
solution containing Coomassie brilliant blue R-250 dye which binds with proteins (Sambrook et al., 1989).
2.2.5.1 Preparation of SDS-PAGE gel
The polyacrylamide gels are prepared by copolymerization of acrylamide and bis-acrylamide (“bis,” N,N'-methylene-bisacrylamide). The copolymerization reaction is basically a vinyl addition reaction initiated by a free radical-generator viz.
Ammonium per sulphate (APS) in presence of N,N,N',N'-tetramethylethane-1,2- diamine (TEMED) which acts as a catalyst (Chrambach, 1985). The components of SDS-PAGE are acrylamide solution 30% (w v-1), resolving gel buffer (Tris-HCl, pH 8.8), a stacking gel buffer (Tris-HCl, pH 6.8), SDS 10 % (w v-1), APS 10% (w v-1), TEMED, sample loading buffer (pH 6.8) and electrophoretic running or tank buffer comprising Tris glycine and SDS, pH 8.3-8.5. The composition of each component of SDS-PAGE gels and buffers are described below in Sections 2.2.5.2-2.2.5.6.
2.2.5.2 Preparation of acrylamide solution
0.8 g of bis-acrylamide was weighed and transferred into an amber colour bottle and dissolved in 50 mL of ultra-pure deionized water (18 MΩcm, Millipore, Milli-Q water purification system) on a magnetic stirrer (IKA, C-MAG HS7). After completely dissolving bis-acrylamide, 29.2 g of acrylamide was added to it to make 30% (w v-1) acrylamide solution and stirred on a magnetic stirrer. The final volume was adjusted to 100 mL with ultra-pure water by keeping the measuring cylinder (100 mL) wrapped with aluminium foil as acrylamide is light sensitive. The acrylamide solution was then filtered (Whatman No. 1) under dark condition and stored at 4°C.
2.2.5.3 Polymerization of SDS-PAGE gel
The resolving gel and stacking gels were prepared following protocols from Sambrook et al. (1989) using the composition as described in Tables 2.2.2 and 2.2.3 given below. The resolving gels were prepared by adding all the components in the order as mentioned in Table 2.2.2, in a 25 mL beaker, by keeping acrylamide concentration to 12% (w v-1) for GH5 cellulase and GH43 hemicellulase hemicellulase (α-L-arabinofuranosidase). Similarly, the stacking gel was prepared by dissolving all the components mentioned in Table 2.2.3. The acrylamide concentration in the stacking gel was kept at 4% (w v-1).
Table 2.2.2 Composition of components for preparation of resolving gel (12%).
Components Volume (mL)
Acrylamide solution (30%, w v-1) 4.0
Deionized water 0.7
SDS (10%, w v-1) 1.0
Glycerol (50%, v v-1) 1.0
1.5 M Tris-HCl (pH 8.8) 3.3
APS (10%, w v-1) 0.1
TEMED 0.01
Table 2.2.3 Composition of components for preparation of stacking gel (4%).
.
Components Volume (mL)
Acrylamide solution (30%, w v-1) 0.7
Deionized water 2.8
SDS (10%, w v-1) 0.5
0.5 M Tris-HCl (pH 6.8) 1.0
APS (10%, w v-1) 0.05
TEMED 0.005
2.2.5.4 Preparation of SDS-PAGE running buffer
The SDS-PAGE gels were run using a 1x running or tank buffer prepared from the 5x stock solution as described below in Table 2.2.4. 15.14 g of Tris free base and 94 g of glycine were dissolved in 800 mL of deionized water. To this 50 mL of 10% (w v-1) SDS was added and the final volume was adjusted to 1 litre. The final pH of the buffer was adjusted to 8.3. The 5x buffer was filtered (Whatman, Filter No. 1) and stored at 4°C.
Table 2.2.4 Composition of 5x Tris-Glycine, running or tank buffer.
Components Final concentration (5x buffer)
Tris base 0.125 M
Glycine 1.25 M
SDS 0.5 % (w v-1)
2.2.5.5 Preparation of sample buffer
5x sample loading buffer was prepared by dissolving the components while keeping the concentration of components as described in Table 2.2.5 and the pH of the buffer was adjusted to 6.8 (Laemmli, 1970). The components were dissolved in the order as mentioned in Table 2.2.5 to make 5x sample buffer. However, the final concentration while loading to a SDS-PAGE gel was always kept to 1x by mixing 4 volumes of sample (protein) with 1 volume of 5x sample buffer. In case of 5x sample loading buffer for non-denaturing SDS-PAGE β-mercaptoethanol was not added.
Table 2.2.5 Composition of 5x sample loading buffer.
Components Final concentration (5x buffer)
Tris-HCl (pH 6.8) 62.5 mM
Glycerol 20.0 (%, v v-1)
SDS 2.0 (%, w v-1)
Bromophenol Blue 0.025 (%, w v-1)
β-mercaptoethanol 5.0 (%, w v-1)
2.2.5.6 Preparation of staining and destaining solutions
The proteins on the SDS-PAGE gel were visualized using a staining solution that contained Coomassie Brilliant Blue (CBB) R-250 dye, which is a disulfonated triphenylmethane (Fig. 2.2.2). The CBB R-250 dye (detection range of 100-1000 ng of protein) formed a non-covalent complex with proteins, based on a combination of Van der Waals forces and electrostatic interactions (Neuhoff et al., 1985). The negatively charged anionic form of the dye is stabilized by formation of a blue colour protein-dye complex which may then be seen on gel (Meyer and Lambert, 1965). The staining solution (100 mL) was prepared by dissolving 250 mg or 0.25% (w v-1), of CBB R-250 dye in 50 mL of deionized water in an amber colour bottle by keeping on a magnetic stirrer for overnight. The solution was filtered (Whatman, Filter No. 1), then 40 mL of methanol and 10 mL of glacial acetic acid were added to finally make the ratio 5:4:1 (deionized water : methanol : glacial acetic acid). The destaining solution was prepared by mixing deionized water: methanol: glacial acetic in 5:4:1 ratio. The gels were destained by immersing in destaining solution with gentle shaking and change of buffer every 30 min, until the protein bands were clear.
Fig. 2.2.2 Chemical structure of Coomassie Brilliant Blue dye R-250 showing