2. Materials and Method
2.5 Biochemical experiments
2.5.3 Western blot (WB) technique
Western blot is a commonly used technique in molecular and cell biology fields. Its aim is to identify particular protein from a mixture of proteins. In addition, this technique involves three steps: the first one is the separation of proteins by their size, the second one is the transformation of the separated proteins to a solid supporter and the last step is the marking of the target protein by specific primary and secondary antibodies for visualization, (Mahmood and Yang, 2012).
Before performing this technique, a specific amount of proteins (γ0 g) was calculated from the concentrations that were obtained from the Bradford assay in order to know the amount of proteins that must be taken from each sample.
The first step for carrying out this technique is the preparation of sample lysate. Which contained 4 l of 1x SDS sample buffer, β l of LDTT, the calculated amount of proteins for each sample and D.W (to complete the final volume to β0 l), all of the components were mixed in 0.2 ml Eppendorf tube. Then mixture was centrifuged at 2.5 RPM for 5 min and placed in the thermocycler, heated at 70 C° for10 min, then stored at -80 C° for the electrophoresis step.
The second step was the preparation of polyacrylamide gel electrophoresis. It consists of two parts: the first part is the (10%) resolving gel, which contains: H2O 2ml, 30%
Acrylamide mix 1.7ml, 1.5M Tris (PH 8.8) 1.3ml, 10% SDS 0.05ml, 10%APS 0.05ml and TEMED 0.02ml. In addition, the second part is the stacking gel which consist of:
H2O 1.4ml, 30% Acrylamide 0.33 ml, 1.5M Tris (PH 6.8) 0.25ml, 10% SDS 0.02ml, 10%APS 0.02ml and TEMED 0.002ml.
The components of polyacrylamide gel electrophoresis were prepared as follow:
-1.5 M Tris-HCL, pH8.8: Tris base 27.23 g, Deionized water 80 ml. Then the pH was adjusted to 8.8 with 6N HCL, then deionized water was added up to 150 ml.
-1M Tris-HCL: Tris base (Giotto biotech) 24.448 g, deionized water 60 ml. Then the pH was adjusted to 6.8 with HCL, then deionized water was added up to 150ml.
-10%APS (Ammonium persulfate): APS 10g and D.W 100ml.
-10% SDS (Bio Rad Laboratories): SDS 10g and D.W 100ml.
-30%Acrylamide (10ml): 40%Acrylamide 7.5ml and DW 2.5ml.
Following polyacrylamide gel electrophoresis preparation, the protein electrophoresis was performed using the Mini-PROTEAN® Tetra Cell apparatus (Bio Rad). Gels were rinsed in1x running buffer using Pasteur pipette. Then the combs were removed, after that they were fixed in the casting stand, placed in the mini tank containing 1x running buffer.
Furthermore, β0 l from each sample and 5 l from the protein marker mix were loaded into the gel and the electrophoresis machine was turned on, the voltage was adjusted to 140 mV for 1h and 40 min approximately.
After the electrophoresis has finished, the gels were transferred into nitrocellulose paper. In addition, the filter papers and the sponges were soaked in 1x transfer buffer, also the nitrocellulose membrane were soaked in D.W first then in the 1x transfer buffer.
Sequentially the sandwich for the transfer was prepared as follow: for each single gel, first the spongy was placed on the transfer cassette over it the wetted filter paper, the gel, wetted nitrocellulose membrane, wetted filter paper and finally wetted spongy. After that air bubbles that lied between any of these layers were removed. Then the wet transfer system was applied according to the Bio Rad instructions.
Moreover, after the transfer has finished the nitrocellulose membranes were stained with commercial Ponceau red solution (0.1 % (w/v) in 5% (v/v) acetic acid) for 30 seconds to visualize the protein bands. Then the stained nitrocellulose membranes were washed by D.W to remove excess Ponceau red stain, scanned by a scanner. Furthermore, the nitrocellulose membranes were placed in plastic boxes for blocking by 5% non-fatty dry milk in 1x PBS-T for 1h. Following the blocking step, membranes were incubated with the specific primary antibody (4HNE 1:1000, for bulbs and plasma samples and CYPA1A11:
1000 for bulbs samples only), overnight at 4 C°. On the following day, membranes were placed in the laboratory agitator at room temperature at a speed of 90 RPM, washed three times with 1x PBS-T for 10 minutes each. Then were incubated with the suitable HRP-conjugated secondary antibodies which were anti-goat (1:5000) for 4HNE antibody (for
OB and plasma samples), and anti- rabbit (1:10000) for Cyp1A1 antibody, (for OB samples only). Moreover, the membranes were washed three times with 1x PBS-T for 10 min each, and with PBS 1x for 15 min. After the washing steps, membranes were incubated in the dark room with Western Lightning® Chemiluminescence Reagent Plus system substrate, by mixing equal volumes of enhanced luminol reagent (1ml) and the oxidizing reagent (1ml) for approximately 5 min. Then the membranes were placed in black box covered with plastic paper and exposed to autoradiography film X-ray in the dark room. In addition, the developed films were lined up in the correct orientation and the molecular weight (MW) ladder bands were marked into the film. Then the developed films were scanned for the quantitative analysis.
All olfactory bulbs membranes were then washed with PBS-T three times 10 min each, then with PBS for 15 min, in the laboratory agitator at room temperature at a speed of 90 RPM. After that membranes were incubated with B-Actin antibody (dilution 1:50000) for 1 h, washed with PBS-T three times 10 min each, then with PBS for 15 min, after that membranes were incubated in the dark room with Western Lightning®
Chemiluminescence Reagent Plus system substrate as described above.
- Preparation of Western blot reagents and Antibodies:
- Ponceau S staining solution: 0.1% (w/v) in Ponceau S stain in 5%(v/v) acetic acid:
Ponceau S1g, acetic acid 50 ml and D.W up to 1 L.
-1X Running Buffer: Tris Base 3g, Glycine (BDH laboratory) 14.3g, SDS 1g and D.w up to 1000ml.
-1%Transfer Buffer: Tris Base 3g, Glycine 14.3g, Methanol 200ml and D.w up to 1000ml 5% Milk solution: Milk 5g and 1x PBS-T 100ml.
-10% PBS: H Cl .200g, KH2PO4 2g, NaCl 80g, Na2HPO4 anidro 11.5g -1%PBS: 10% PBS 100ml and D.W 900ml.
-1%PBS-T: 10% PBS 100ml, D.W 900ml and Tween 1ml.
-1%SB (sample buffer): 5%SB 1ml and 2-mercaptoetanolo 0.05ml.
-4HNE antibody (Biorad-1:1000 / 6ml): PBS-T 5.4ml, 5%milk 0.6 ml and 4HNE 0.006 ml -CYP1A1 antibody (Thermo fisher -1:1000- 6 ml): PBS-T 5.4ml, 5%milk 0.6 ml and CYP1A1 0.006 ml.
-actin antibody (Biorad laboratory-1:50000-10ml): PBS-T 9ml, 0.5%milk 1ml and B-actin antibody 0.002ml.
-Anti-Goat antibody (Biorad- 1:5000- 10ml): PBS-T 9ml, 0.5%milk 1ml and Anti-Goat antibody 0.002ml.
-Anti-Rabbit antibody (Biorad-1:10000-10ml): PBS-T 9ml, 0.5%milk 1ml and Anti-Rabbit antibody 0.001ml.