SDS-PAGE (Sodium dodecyl sulphate-polyacrylamide gel electrophoresis) FOLLOWED BY WESTERN BLOTTING USE TO DETERMINE THE RESPONSE OF HT29, SW620 AND HCT116 TO ETOPOSIDE, CISPLATIN AND PACLITAXEL TREATMENT INTRODUCTIONSDS-PAGE/Western Blotting combination is one of the most commonly used laboratory techniques to determine the presence and level of expression of a specific protein in a mixture of proteins.SDS-PAGE is the electrophoresis method that is used to separate a complex mixture of proteins by using electric current which applied to the gel. The first step involves protein denaturation by SDS which acts as a powerful detergent. This step is important to make sure that the movement of the proteins which then placed on top of gel is determined by their molecular weight and not by their secondary structure.The second step is the western blotting to determine the expression and level of specific proteins. This is done by using specific antibodies which has been probed to the proteins and these specific antibodies will bind to antibodies which can be detected using the enhance chemiluminiscence (ECL). This procedure is used to determine protein subunit composition and fractionation.1 Proteins are placed on top of a gel of cross-linked acrylamide and a current is applied to the gel. Thousands of polypeptides are denatured and separated and when used in conjunction with blotting methods, this combination method has been proved to be one the most powerful means available for protein analysis.2 Western blot data also is widely used method and efficient tool to determine and compare the expression of specific proteins in various cells and tissues3. This method can be summarized in (Figure 1)Figure 1: Schematic diagram of typical western blot. The typical western blot requires gel electrophoresis to separate proteins based upon molecular mass with subsequent transfer of the proteins from the gel to a protein binding membrane. The membrane is then incubated with the primary antibody to the target protein and the primary antibody is detected by a tagged secondary antibody. The tagged secondary antibody catalyzes an enzymatic reaction with the substrate which can be detected by film or a digital imager.The reason why this method is highly sensitive and very specific is due to two main factors: 1) Proteins are separated according to different sizes, charge and conformation by gel electrophoresis. 2) The specific interaction between antibody and antigen. Target protein in complex mixtures containing > 100,000 different proteins can be detected by using this selective nature of the specific antibody.3 METHODS AND MATERIALSSteps in the process proceed as follows:1)      Preparation of cell/tissue sample2)      Lysing the tissue/cell sample3)      Determining the protein concentration of the samples4)      Performing SDS-PAGE of a suitable amount of protein5)      Staining gel to reveal protein bands and/or6)      Transfer of proteins to nitrocellulose paper-western blotting7)      Probing of the transferred (blotted) proteins with antibodies and revealing their location of ECL8)      Stripping and re probing with a loading control antibody1)      Preparation of cell samplesHT29, SW620 and HCT116 colon cancer cells were grown as adherent monolayers. Cell lines were treated with 10µM concentrations of etoposide, cisplatin, and paclitaxel for 48 hours.2) Detergent lysis of cell samplesThe cells were detached from plastic with trypsin/versene, washed in serum-free medium by centrifugation (1200rpm for 5 minutes) and the washed cell pellets lysed for 30 minutes on ice with lysis buffer containing protease inhibitors. A rate of 2-10.106 cells per ml of lysis buffer is normal. Lysates are centrifuged for 10 minutes at 4oC at 10-15,000g to remove insoluble material. The supernatant is removed carefully without disturbing the pelleted insoluble material and then aliquoted into smaller volumes (50-100µl) and stored frozen (-20oC). This is to avoid the desructions of proteins caused by repeat freezing-thawing.3) Determining the protein concentration in cell lysates using BioRad DC Assay KitThe Bio-Rad DC Protein Assay is a colometric assay that is used to measure the concentration of proteins in cell lysates.Proteins are released from cells/tissues by treatment with a detergent. The protein in the sample reacts with the copper in an alkaline copper tartrate solution. Folin reagent is subsequently added and reduced by the copper-treated protein. This results in a blue colour, the intensity of which is dependent on the concentration of protein in the lysate. The blue colour reaction has a maximum absorbance at 750nm and a minimum absorbance at 405nm.Absorbance is read on a spectrophotometer and protein concentration in the test samples is calculated from a standard curve constructed from samples of known protein concentration.Reagents and equipmenta)      Flat bottomed 96 well microtiter plateb)      Pipettes and tips for delivering 5µl, 25µl and 200µlc)       Bio-Rad Reagent A (alkaline copper tartrate solution)d)      Bio-Rad Reagent B (dilute Folin Reagent)e)      Bio-Rad Reagent S (sodium dodecyl sulphate)f)        Bovine Serum Albumin (BSA) standardsg)       Samples to be testedMethodPrepare protein standards containing 0, 100, 200, 400, 800, 1200, 1600 and 2000 mg/ml BSAFor each standard provided, pipette 5µl into 3 wells of a 96 well ELISA plate.Pipette 5µl of each test samples into 3 wells of the plateActivate reagent A by adding 20 µl reagent S to 1 ml of reagent AAdd 25 µl of activated reagent A to each well containing standard or sampleAdd 200µl of reagent BAfter 15 minutes read the developing blue colour on a 96-well plate spectrophotometer, measuring optical density at 750nm wavelength.4)Performing SDS PAGEi) BioRad SDS-PAGE equipment was combined with two 4-20% Tris-HCI BioRad precast gels in place.ii) HT29, HCT116 and SW620 cells were treated with 10µM of nil drug, etoposide, cisplatin or paclitaxel (i.e 4 different treatments) for 48 hours and cells were lysed and protein concentrations were already determined and aliquoted on ice at 2mg/mliii) Micro-centrifuge tubes containing 15µl of lysates (which equivalent to 30µg of protein) were prepared for each different cell line, and for each different treatment therefore total of 12 tubes. 15µl of 2x Laemmli reducing loading buffer (Sigma) were added to each sample. These solutions were mixed by gently tapping the tubes, then the tubes were placed for 5 minutes into a heat block which is set to 100oC. This helps the SDS and reducing agent in the Laemmli buffer to denature the proteins.iv) Condensation on the lid is monitored. Centrifuge samples were pulsed to collect samples at bottom of tube; which indicates samples are ready for loading.v) Then by using long-fine pipette tip, 30µl sample mixture was loaded into the wells. Carefully the end of the tip was placed at the bottom of the well to gently expulse the sample, and this needs to be done by making sure there are no air bubbles at the end of expulsion. The first well was marked with molecular weight markers (10µl is enough). These are often pre-stained and can be seen migrating down the gel during electrophoresis.vi) The order that the samples were loaded were recorded clearlyvii) The lid was placed on the gel apparatus and power-pack was connected under the demonstrators directionviii) The gel was runned at 10’ at 120V to let samples concentrate and enter the gel, then for 200V till the bromophenol blue-dye front nears bottom about 30-40 minutes.ix) While gel is running, transfer buffer was prepared for western blotting.4) Staining gels to reveal protein bandsCell Lysis Buffer (Biosource)-          50mM Tris, pH 7.4-          250mM NaCl-          5mM EDTA-          50mM NaF (sodium fluoride-a phosphatase inhibitor)-          1mM Na3 VO4 (sodium othovanadate -a phosphatase inhibitor)-          1% Nonidet P40 (NP40)Add protease inhibitors (Calbiochem; Cocktail Set 1) to 1x strength10x Tris/Glycine/SDS Running buffer stock solution100ml of 10x concentrate was added to 900ml of distilled water to make 1 Litre of working buffer and was thoroughly mixed.So final concentration of a 1x concentration is:-          25mM Tris-          192mM Glycine and 0.1% (w/v) SDS, pH 8.3Transfer Buffer100 ml of 10x Tris/Glycine concentrate buffer stock solution was added to 200ml of methanol and 700ml of distilled water to make 1 Litre of working transfer buffer and thoroughly mixed.So final concentration of 1x solution is:-          25mM Tris-          192mM Glycine-          20% Methanol, pH 8.3 TBS/Tween Wash Buffer4ml of 10% Tween was added to 400ml of Tris buffered saline and were mixed thoroughly to make up a litre.5% Milk Blocking Buffer5% Milk solution: 5gm of milk powder (Marvel) was added to 100ml of wash buffer to make up 200ml6) Transfer of proteins to nitrocellulosei) The gel was switched off and disassembleii) Using the BioRad ‘knife’ provided, the pre-cast gel was openediii) With gloved hands, the gel was carefully lifted and was placed into a small containeriv) Nitrocellulose Membrane and filter papers were soaked together with the sponges in 1x transfer bufferv) Blotting cassette was placed onto a clean surface with the dark grey side downvi) One pre wetted sponge was placed on the grey side of the cassettevii) A sheet of filter paper was placed on the sponge and air bubbles were removed by rolling the pipetteviii) With gloved hands, gel was lifted and placed onto the filter paper.ix) To make sure that the nitrocellulose is not contaminated with proteins on the fingers, with gloved fingers, the pre wetted nitrocellulose membrane was lifted and placed on top of the gel carefully and gently rolled to remove air bubbles.x) The second piece of filter paper was placed onto the membrane followed by other sponge.xi) The cassette was firmly closed whilst being careful not to disturb the gel and the filter paper.xii) The cassette was locked and closed with white latchxiii) The cassette was placed in the blotting module that was provided and this was repeated the same for the other cassettexiv) The frozen ice cooling unit was placed into the tank and the tank as completely filled with transfer bufferxv) Lid was placed and cable was plugged into the power pack and the blot was runned at 100V for one hour to transfer proteins.xvi) While the blot is transferring, blocking buffer was prepared (4% Marvel)xvii) Upon completion of the run, blotting sandwich was disassembledxvii) The membrane is removed and 10ml off the blocking buffer was placed in the dishes provided.xix) The dishes were clearly labelled. The dishes were tightly sealed with cling film to prevent any evaporation. The dishes were left at 4oC till the next session (usually 1-2 weeks)7)Probing of the transferred (blotted) proteins with antibodies and revealing their location with ECLi) Clearly 50ml falcon tube was labelled with the name of the primary antibody and 5ml of fresh blocking solution was added.ii) 5µl of the primary antibody provided was added to the blocking solution in the Falcon to give a final dilution of 1:1000 and well mixed.There are two antibodies:a)      Mouse monoclonal antibody to active + pro caspase 3 (Supplier: Abcam)b)      Monoclonal Mouse anti-human p53 protein, Clone DO-7 (Supplier: Dako)iii) Membrane was removed from the blocking solution which was discarded later, and the membrane was inserted carefully into the 50ml centrifuge tubeiv) The tubes were left on a roller for 1 hour at room temperature.v) The TBS/Tween solution was preparedvi) The membrane was carefully removed from the tube and was transferred to a dish containing 10ml of TBS/Tween (wash buffer) and was placed onto an orbital shaker-rotate.vii) It was then washed with 3 changes of the wash buffer, 5 minutes each.viii) Another TBS/Tween was prepared and clearly labelled, by adding 6ml of the milk-blocking buffer in 50ml tube and 2µl of rabbit anti-mouse HRP (Dako) was added and well mixed.ix) The tube was left on the roller for 45 minutes at room temperature.x) The membrane was carefully removed from the tube and was transferred to a dish containing 10ml of TBS/Tween (wash buffer) and was placed onto an orbital shaker and washed x3 as above.During the last wash, Enhanced Chemiluminescence reagent was prepared in a bijou by mixing 2ml of reagent 1 with 2ml of reagent 2.xi) A clean sheet of cling film was placed on the bench and was carefully done to avoid any creases.xii) Carefully, the membrane from the wash buffer was transferred onto the cling filmxiii) 2ml of the ECL mix was added onto each membrane by making sure the entire surface is covered.xiv) It was left for one minute and quickly ECL was drained but by making sure the membrane remains wet.xv) The membrane was transferred onto an autoradiography cassette as instructed. Blots were covered with cling film to keep damp. The cassette was then closed after making sure all air bubbles were carefully removed.xvi) The membrane to X-ray film was exposed in a dark room for one minute.xvii) Fresh film was re-exposed if necessary using longer or shorter exposure times as appropriate.8)Stripping and re-probing with a loading control antibodyi) The membrane was washed in the wash buffer as aboveii) After the final wash, the membrane was transferred to stripping buffer (Chemicon International) at 1:10 dilution and was placed on a shaker for 15 minutesiii) The membrane is washed in wash buffer as aboveiv) The membrane was blocked in the block buffer for 30 minutesv) The blot was transferred to a 50ml tube containing an antibody specific for an appropriate loading control protein expressed by the cells e.g HSC70RESULTSBased on this result, cell lines for HT29, it was evenly exposed regardless to any drug treatment even with no drug. There are bands that shows 53kDa corresponding to the size of p53. This suggests that p53 are endogenous cells.In contrast, SW620 shows different type of pattern of expression. As we can see, the first three darker bands from the left corresponds to nil drug, etoposide and cisplatin respectively. There is no band expressed with taxol treatment which suggests that, etoposide and cisplatin induce expression of p53 but weaker band so less expression of p53 which indicates SW620 shows no respond to taxol treatment.For HCT116 only etoposide induced expression of p53 and there are no bands expressed in the other drug treatment. Hence this suggests that HCT116 shows responds to etoposide treatment.For caspase 3, the cell lines in HT29 and SW620 show same intensity of darker bands regardless of any drug treatment. This indicates that all treatment induced expression of caspase 3 in those two cell lines. In contrast, for HCT116, there is weaker band which correlates to 17kDa which shows cleaved caspase 3 are present in HCT116. Etoposide treatment induce expression of cleaved caspase 3 in HCT116 which suggests that HCT116 cell lines show lack of respond to etoposide treatment.DISCUSSIONSColorectal cancer is a commonly observed malignant cancer. As we know, aetiology of colon cancer includes changes in genetic mutation and changes within cell communication pathway. For this experiment, Western blotting is used to determine how the cell lines respond to above drug treatments.One of the most frequently reported gene that can cause cancer is p3 gene mutation.4,5 Gene p53, which is located on chromosome 17p13,6,7. Different literatures showed that these gene changes had been linked with different types of cell lines of cancer diseases. Three top worldwide cancers such as lung,8,9 breast,10 and colon11 are associated with p53 gene mutation. This changes in p53 gene will convert normal p53, a tumour suppressor gene, to a dominant oncogene. 12-14 For colon carcinomas, more than 75% of colon carcinomas are usually associated with loss of heterozygosity of that locus of p53 gene.15-17Some gene products are responsible as apoptotic effectors proteins and anti-apoptosis regulators.18 Caspases are a gene family that acts in a cascade manner and caspase 3 is one of the final effectors leading to apoptosis. 19Heat shock cognate 71 kDa protein (HSC70), is a member of the heat shock protein 70 family (HSP70) and associates with nascent CFTR on the ribosome20. Thus, cessation of this interaction correlates with cessation of CFTR ubiquitination21 It is a constitutively expressed chaperone protein and is involved in diverse cellular processes including protein folding and protein degradation. Since these genes are constitutively expressed, it also known as housekeeping genes which means it is act as control genes for this experiment.Western blotting is a technique that involves the separation of proteins by gel electrophoresis, and few other steps as described above. It plays a role as a routine method for protein analysis that depends on the specificity of antibody-antigen interaction, therefore it is useful for the qualitative or semiquantitative identification of specific proteins and their molecular weight from a complex mixture.22However, like many other lab methods, Western blotting also have few disadvantages. The main disadvantage of Western blotting is that this technique requires a specific antibody to a target protein. Due to lack of specific antibodies, many protein targets cannot be studied.23 Some critical parameters which are needed to reproduce the results were not reported in few studies. These parameters include the amount of proteins loaded, the blocking solution and conditions used, the amount of primary and secondary antibodies used, the antibody incubation solutions, the detection method and the quantification method utilized.23In order to improve conventional Western blotting, recent discovery has led to use of single-cell western blotting (scWB). This new improvised technique is useful for protein targets that lack selective antibodies (e.g isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of polyacrylamide (PAG). This technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine. 24REFERENCES:1)      https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/9780471729259.mca03ms222)      David E.Garfin, Chapter 29 One-Dimensional Gel Electrophoresis. Methods in Enzymology. Volume 463, 2009, Pages 497-513 3)      Rajeshwary Ghosh,  Jennifer E. Gilda, and Aldrin V. Gomes. The necessity of and strategies for improving confidence in the accuracy of western blots. Expert Rev Proteomics. 2014 Oct; 11(5): 549–560.4)      Harris AL: Editorial: Mutant p53-the commonest genetic abnormality in human cancer? J Pathol 1990, 162:5-65)      Nigro JM, Baker SJ, Preisinger AC, Jessup JM, Hostetter R, Cleary K, Bigner SH, Davidson N, Baylin S, Devilee P, Glover T, Collins FJ, Weston A, Modali R, Harris CC, Vogelstein B: Mutations in the p53 gene occur in diverse human tumour types. Nature 1989, 342:705- 7086)      Isobe M, Emanuel BS, Givol D, Oren M, Cvoce CM: Localization of the gene for human p53 tumour antigen to band 17p13. Nature 1986, 320:84-857)      McBride OW, Merry D, Givol D: The gene for human p53 cellular tumor antigen is located on chromosome 17 short arm (17p13). Proc NatI Acad Sci USA 1986, 83:130-1348)      Iggo R, Gatter KC, Bartek J, Lane DP, Harris AL: Increased expression of mutant forms of p53 oncogene in primary lung cancer. Lancet 1990, 335:675-6799)      Chiba I, Takahashi T, Nau MM, D’Amice D, Curiel DT, Mitsudomi T, Buchhagen DL, Carbone D, Piantadosi S, Koga H, Reissman PT, Slamon DJ, Holmes EC, Minna JD: Mutations in the p53 gene are frequent in primary, resected non-small cell lung cancer. Oncogene 1990, 5:1603-161010)  Prosser J, Thompson AM, Cranston G, Evans HJ: Evidence that p53 behaves as a tumor suppressor gene in sporadic breast tumors. Oncogene 1990, 5:1573-157911)  Baker SJ, Fearon ER, Nigro JM, Hamilton SR, Preisinger AC, Jessup JM, Van Tuinen P, Ledbetter DH, Nakamura Y, White R, Vogelstein B: Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science 1989, 244:217-22112)  Herskowitz l: Functional inactivations of genes by dominant negative mutations. Nature 1987, 329:219-22213)  Hinds P, Finlay C, Levine AJ: Mutation is required to activate the p53 gene for cooperation with the ras oncogene and transformation. J Virol 1989, 63:739-74614)  Lane DP, Benchimol S: p53: oncogene or anti-oncogene? Genes Dev 1990, 4:1-815)  Baker SJ, Fearon ER, Nigro JM, Hamilton SR, Preisinger AC, Jessup JM, Van Tuinen P, Ledbetter DH, Nakamura Y, White R, Vogelstein B: Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas. Science 1989, 244:217-22116)  Baker SJ, Preisinger AC, Millburn Jessup J, Paraskeva C, Markowitz S, Willson JKV, Hamilton S, Vogelstein B: p53 gene mutations occur in combination with 17p allelic deletions as late events in colorectal tumorigenesis. Cancer Res 1990, 50:7717-772217)  Fearon ER, Vogelstein B: A genetic model for colorectal tumorigenesis. Cell 1990, 61:759-76718)  Elmore S. Apoptosis: A review of programmed cell death. Toxicol Pathol. 2007;35(4):495–516. doi: 10.1080/0192623070132033719)   Lee G, Kim J, Kim Y, Yoo S, Park JH. Identifying and monitoring neurons that undergo metamorphosis-regulated cell death (metamorphoptosis) by a neuron-specific caspase sensor (Casor) in drosophila melanogaster. Apoptosis. 2018;23(1):41–53. doi: 10.1007/s10495-017-1435-6.20)  Meacham, G. C., Lu, Z., King, S., Sorscher, E., Tousson, A. & Cyr, D. M. (1999) EMBO J. 18, 1492–1505.21)  Fuller, W. & Cuthbert, A. W. (2000) J. Biol. Chem. 275, 37462–3746822)  Thomas S. Hnasko , Robert M. Hnasko. The Western Blot. ELISA pp 87-9623)  Jennifer E. Gilda, Rajeshwary Gosh, Jenice X. Cheah, Toni M.West, et al. Western blotting Inaccuracies with Unverified Antibodies : Need for a Western Blotting Minimal Reporting Standard (WBMRS) PLoS ONE 10 (8):e0135392. Doi:10.1371/journal.pone.013539224)  Chi-Chih Kang, Kevin A Yamauchi, Julea Vlassakis et al. Single cell-resolution western blotting. Nature Protocols 11, 1508-1530 (2016)