Optimization of antibody concentration and the detection limit of human recombinant CTGF. Senior Honors Thesis Dilan Patel 8824 8548 July 15, 2010 Abstract The purpose of this experiment was to gain an understanding of both the optimal antibody concentration and the limit of detection of human recombinant CTGF. Gel electrophoresis and western blotting were conducted to help produce results. The CTGF concentrations and the antibody concentrations were varied over a mu ltitude of gels. In this experiment, it was determine d that the C terminal region of CTGF b ound optimally to a 1.5 e hinge region of the CTGF bound strongly to all concent ration s of antibody. Finally, the N terminal region of CTGF did not bind as strongly when compared to the C terminal or the hinge region. The optimal antibody concentration for the N terminal region was difficult to determine due the lack of quality in the images. However, it seem ed as though when the antibody concentration wa s increased, the ability to bind to the N terminal CTGF decreased
Dilan Patel 8824 8548 Honors Thesis 2 Fig 1 Modulation of C orneal W ound H ealing Shortly after injury, epithelial cells are activated via both the autocrine system and lacrimal gland. Introduction After suffering trauma in the cornea, a complex cascade known as stromal wound healing occurs The stromal wound healing contains multiple growth factors, cytoki nes, chemokines, and proteases (Fig 1) Directly following the epithelial damage, the process of healing begins via the ini tiation of multiple cytokiness and growth factors including, but not limited to, interleukin 1 (IL 1) 1 tumor necrosis factor alpha 2,3 bone morphogenic proteins 2 and 4 4 epidermal growth factor (EGF) 5 platelet derived growth factor (PDGF) 6 and transforming growth factor beta (TGF 7 All of these mediates are released by either epithelial cells or the lacrimal gland. In normal, non injured corneal stromal tissue quiescent keratocytes exist due to minimal
Dilan Patel 8824 8548 Honors Thesis 3 synthesis of proteins, DNA and RNA. However, upon injury the keratocytes transform rapidly and become activated. This was proven by microarray analysis of rat corneas following p hotoref r active k erato tomy (PRK) which found that levels of 5,885 genes changed during the first 12 days of healing 8. The activated keratocytes play major roles in repairing corneal tissue following injury. Prol iferation and migration of the activated keratocytes begins 12 to 24 hours after epithelial injury 9 The proliferating keratocytes give rise to activated keratocytes, fibroblasts, myofibroblats which reproduce the depleted stroma 10 14 After 1 to 2 weeks, myofibroblast a ppear in the epithelial stroma 15 which secrete many growth factors including TGF TGF is a 25 kDa, disulfide linked homodimeric protein that is involved in apoptosis control, angiogensis, wound healing, immune regulation and tumor bio logy. In the wound healing process, TGF monocytes, macrophages and fibroblasts, which contribute to the inflammatory phase of wound hea ling 16 17 TGF also induces the synthesis of collagen by corneal fibroblast which is mediated by connective tissue growth factor (CTGF ) 18 CTGF is a downstream mediate of TGF which promotes cell adhesion and mitogenesis in both fibroblasts and endothelial cells and stimulates cell migration in fibroblasts. In angiogenic tissues as well as in atherosclerotic plaques CTGF exists suggesting a possible role in the regulation of vessel growth during development, wound healing and vascular disease. CTGF is a 38 kDa, single chain cysteine rich protein that is secreted through t he Golgi apparatus 19 The overall structure of CTGF can be separated into three major segments : the N terminal domain, the hinge region, and the C terminal domain (Fig. 2 ). CTGF has been implicated in numerous biological activities, including stimulation o f cell migration, proliferation, extracellular matrix synthesis, adhesion,
Dilan Patel 8824 8548 Honors Thesis 4 survival, differentiation, and apoptosis 20 21,22 The different domains of CTGF are said to have mutually exclusive biological functions, proliferation and differentiation. Grotendorst and Duncan exemplified that the C terminal fragment o f CTGF stimulates proliferation while the N terminal fragm ent stimulates differentiation 23 (Fig 3) Fig 2 CTGF P redicted Cleavage S ites, M ajor D omains and P rotein M odules The following CTGF protein depicts the predicted cleavage site, the N terminal domain, the C terminal domain and the hinge region. Fig 3 Corneal I njury C ascade of E vents Following corneal injury, a cascade of events occur in a short period of time. In addition proteolytic cleavage leads to the two different CTGF fragments.
Dilan Patel 8824 8548 Honors Thesis 5 The overall aim of this study was to determine the role of the different CTGF fragments throughout wound healing. However, in order to understand the overall roles, many smaller projects were devised to gain an understanding on h ow CTGF can both be measured and trace d In addition, a method had to be derived to help evaluate the ratio of N terminal and C terminal CTGF fragments during the corneal wound healing both in vivo and in vitro Throughout the experiment, protein gel electrophoresis, western blotting, and c ell culturing were carried out. The specific purpose of this thesis was to determine the limit of detection of CTGF as well a s to optimize the minimum amount of antibody required to bind to CTGF. The antibodies that were used were monoclonal meaning that they were created from a single cell line and were made to respond to diffe rent regions of the CTGF protein. Experimental Procedure Gel Electrophoresis : The gels used in this procedure were ready made and were manufactured by NuPAGE Novex ( The specific product is called Bis Tris Mini Gels ). The gels must be stored at +4 C and only taken out when ready to load the wells. Prior to loading, t he samples have to be prepared by adding LDS sample buffer (4X) and reducing agent (10X) The LDS sample buffer contains c oomassie G250 and phenol red which are both used as tracking dyes instead of the r egularly used bromophenol blue Each well contained a sample volume of LDS sample buffer (4X) and reducing agent (10X) volume of The sample in this experiment was recombinant human CTGF with a stock concentration of 100 ng/ Once the samples were made, the running buffer was prepared by mixing 25 mL 20 X SDS Running Buffer with 500 mL deionized water. The samples were then loaded into the ir respective wells and a precision plus
Dilan Patel 8824 8548 Honors Thesis 6 protein dual color standard was also used. T he entire cell was then taken to an electric sour ce and plugged in. The gels were run for approximately 35 minutes at 200 volts beginning with 110 mA and ending with 70 mA. After the electrophoresis was comple te, the electrodes were removed and t he gel cassette was cracked open The gel was cut from the casing and was ready for the i B lot Dry Blotting Transfer procedure. iBlot Dry Blotting System : The protein information on the gel is blotted onto a PVDF membrane in about 7 minutes. Figure 4 depicts how the convenient transfer system works: Once the gel was cut out of the cassette, it was placed on the bottom PVDF membrane which was topped with filter paper which was then topped with a conducting membrane and a sponge. Once the 7 minute transfer was complete everything but the PVDF membrane was disposed of Fig 4 Invitrogen iBlot Dry Blotting System This system removes the need for layered filter paper and buffer tanks. The top and bottom stacks contain the required buffers. The bottom stack also includes an integrated 0.2 PVDF membrane.
Dilan Patel 8824 8548 Honors Thesis 7 appropriately. The PVDF membrane now contained the proteins of interest and was ready for the western blot procedure. Western Blotting : The PVDF membrane was washed in methanol, deionized water and 1X PBS for 1 minute in each. F ollowing the washes, each gel was placed in 5 mL of Odyssey Blocking Buffer for approximately 1 hour. The blocking buffer often yields higher and more consisten t sens itivit y. Following the 1 hour blocking period, the primary antibody was added and was left on a shaker overnight in the 4 C cooler Once the primary antibody wa s removed, the mem brane was washed 4 times with 5 mL of 0. 1% Tween 20 to PBS mixture. After the light sensitive secondary antibody was added, the membrane was left to shake for approximately 2 hours. Following the 2 hour period, the membrane was again washed with 5 mL of 0. 1% Tween 20 to PBS mixture Subsequent to the wash, the membrane was ready for imaging using a fluorescence signal detector. The results were then analyzed for presence, size, and quantity. Calculations Most o f the experimental data was qualitative ; however, calculation of the varyi ng antibody concentra tion required the dilution equation: M 1 V 1 = M 2 V 2 (1) The variable M 1 refers to the concentration of species 1 and V 1 refers to the volume of species 1. A sample calculation is shown below:
Dilan Patel 8824 8548 Honors Thesis 8 Equation #1 M 1 V 1 = M 2 V 2 M 1 = 8.49 V 1 = Unknown M 2 = 0.50 V 2 = 1.5 0 mL ( 8.49 ) ( V 1 ) = ( 0.50 ) ( 1.5 0 mL ) V 1 = 0.0 9 antibody (C terminal) Data and Results Each gel was loaded the same way and the specific order is shown below : Following each gel electrophoresis, a western blot was conducted. The western blot required three different monoclonal primary antibodies which were developed in our lab. The different antibodies targeted specific regions of the CTGF protein. Thus, the sto ck concentration for each antibody was used to calculate the required volumes for each trial. A total of 9 trials were conducted to determine which antibody concentration was optimal. Antibody Additions Stock Concentrations Desired Concentration Required Volume C terminal 8.49 0.50 0.0 9 1.00 0.1 8 1.50 0.2 7 Hinge Region 2.39 0.50 0.31 1.00 0.6 3 1.50 0.94 N terminal 3.60 1.50 0.6 3 5.00 2.08 10.00 4.1 7 Well Number 1 2 3 4 5 6 Sample Dual MW Std. 100 ng CTGF 50 ng CTGF 25 ng CTGF 12.5 ng CTGF 0 ng CTGF Table 1. The gel loading specifications Table 2 Primary antibody volume requirements
Dilan Patel 8824 8548 Honors Thesis 9 The fi rst set of western blots depicted the C terminal antibodies and the corresponding antibody concentrations. The next set of western blots depict ed the hinge region antibodies and the corresponding antibody concentrations. Fig 5 C terminal antibody. Primary antibody binds to the CTGF protein around 38kDa Fig 6. Hinge region antibody. Primary antibody binds to the CTGF protein around 38kDa kDa 150 100 75 50 37 25 kDa 150 100 75 50 37
Dilan Patel 8824 8548 Honors Thesis 10 The next set of western blots depict ed the N terminal antibodies and the corresponding antibody concentrations. The brightness and co ntrast were adjusted to help observe the CTG F band at the 38 kDa location. The images were cut into small sizes in order to fit into the corning 35mm culture dishes which explains the different shapes Fig 7. N terminal antibody. Primary antibody binds to the CTGF protein around 38kDa
Dilan Patel 8824 8548 Honors Thesis 11 Conclusion The major results of the experiment were qualitative and serve as a foundation for future procedures In this experiment, it was determine d that the C terminal region of CTGF bound optimally to a 1.5 it also bound to concentrations as low as 0.5 The hinge region of the CTGF bound strongly to all concentration s of antibody. Finally, th e N terminal region of CTGF did not bind as strongly when compared to the C terminal or the hinge region. The optimal antibody concentration for the N terminal region was difficult to determ ine due the lack of qu ality in the images. However, it seems as though wh en the antibody concentration was increased, the ability to bind to the N terminal CTGF decreased The monoclonal antibodies were made by the College of Medicine at the University of ecifications detailing optimal binding conditions. On the other hand, c ommercially available monoclonal anti CTGF presents images that exemplify optimal binding and is shown here: Fig 8. O ptimal binding of CTGF Commercially available antibody binds to the CTGF protein around 38kDa
Dilan Patel 8824 8548 Honors Thesis 12 The images that captured during the experi ment show that CTGF binds s imilar ly to the commercially available anti CTGF t hus verifying that the lab made antibodies are successful at binding CTGF. The two goals in this experiment were to determine both the optimal antibody concentration and the limit of detec tion of human recombinant CTGF. However, a shortcoming of this experiment was determining the limit of detection The images that were captured did not have any indication of a standard curve and therefore figures 5, 6 and 7 were modified to only show the pertinent data. The reason for this shortcoming could be as simple as the limit o f detection is in fact 100 ng; however, the more logical reason is that the serial dilution was conducted incorrect ly A myriad of things could have occurred including: incorrect pipette volumes, not enough sample mixing, or bad stock solution. In the next few weeks this experiment will be conducted again with emphasi s on the varying amounts of CTGF. This project aids in future experimentation because it will optimize certain protocols that deal with the s imilar antibodies. Knowledge of the limit of detection of CTGF will help minimize waste and reduce cost within the lab. Overall, u nderstanding how CTGF behaves in vitro and in vivo will help in the development of gene therapy Gene therapy is used to alter gene expression by interfering with either cytosolic mRNA or translated protein. In terms of wound healing, targeting and controlling either or both TGF and CTGF at the molecular level will allow for both a possible reduction in corneal haze and a successful project.
Dilan Patel 8824 8548 Honors Thesis 13 References 1. R. R. Mohan et al., Exp.Eye Res. 65, 575 589 (1997). 2. R. Pellizzari, C. Guidi Rontani, G. Vitale, M. Mock, C. Montecucco, FEBS Lett. 462, 199 204 (1999). 3. R. R. Mohan, R. R. Mohan, W. J. Kim, S. E. Wilson, Invest Ophthalmol.Vis.Sci. 41, 1327 1336 (2000). 4. R. R. Mohan, W J. Kim, R. R. Mohan, L. Chen, S. E. Wilson, Investigative Ophthalmology & Visual Science 39, 2626 2636 (1998). 5. S. E. Wilson, L. Chen, R. R. Mohan, Q. W. Liang, J. Liu, Exp.Eye Res. 68, 377 397 (1999). 6. I. S. Tuominen et al., Exp.Eye Res. 72, 631 64 1 (2001). 7. J. V. Jester, J. Huang, W. M. Petroll, H. D. Cavanagh, Exp.Eye Res. 75, 645 657 (2002). 8. S. Tuli, M. Goldstein, G. S. Schultz, in Cornea J. H. Krachmer, J. M. Mannis, E. J. Holland, Eds. (Elsevier Mosby, Philadelphia, 2005) ,chap. 9. 9. M. V. Netto et al., Cornea 24, 509 522 (2005). 10. M. E. Fini, Prog.Retin.Eye Res. 18, 529 551 (1999). 11. L. Gan, H. Hamberg Nystrom, P. Fagerholm, G. Van Setten, Acta Ophthalmol.Scand. 79, 488 492 (2001). 12. J. L. Andresen and N. Ehlers, Curr.Eye Res. 17, 79 87 (1998). 13. P. O. Denk and M. Knorr, Graefes Arch.Clin.Exp.Ophthalmol. 235, 530 534 (1997). 14. K. Musselmann, B. P. Kane, J. R. Hassell, Exp.Eye Res. 77, 273 279 (2003). 15. S. E. Wilson et al., Arch.Ophthalmol. 119, 889 896 (2001). 16. S. M. W ahl et al., Proc.Natl.Acad.Sci.U.S.A 84, 5788 5792 (1987).
Dilan Patel 8824 8548 Honors Thesis 14 17. A. E. Postlethwaite, O. J. Keski, H. L. Mosses, A. H. Kang, J Exper Medicine 165, 251 256 (1987). 18. T. D. Blalock et al., Invest Ophthalmol.Vis.Sci. 44, 1879 1887 (2003). 19. Y. Chen, P. Segarini, F. Raoufi, D. Bradham, A. Leask, Exp.Cell Res. 271, 109 117 (2001). 20. G. R. Grotendorst, Cytokine Growth Factor Rev. 8, 171 179 (1997). 21. L. F. Lau and S. C. Lam, Exp.Cell Res. 248, 44 57 (1999). 22. C. Ayer Lelievre et al., Mol.Pathol. 54, 1 05 120 (2001). 23. G. R. Grotendorst and M. R. Duncan, FASEB J. 19, 729 738 (2005).
Optimization of antibody concentration and the detection limit of human recombinant CTGF. Senior Honors Thesis Dilan Patel July 15, 2010 Abstract
Dilan Patel 8824 8548 Honors Thesis Fig 1 Modulation of C orneal W ound H ealing Introduction
Dilan Patel 8824 8548 Honors Thesis
Dilan Patel 8824 8548 Honors Thesis Fig 2 CTGF P redicted Cleavage S ites, M ajor D omains and P rotein M odules Fig 3 Corneal I njury C ascade of E vents
Dilan Patel 8824 8548 Honors Thesis Experimental Procedure
Dilan Patel 8824 8548 Honors Thesis Fig 4 Invitrogen iBlot Dry Blotting System
Dilan Patel 8824 8548 Honors Thesis Calculations
Dilan Patel 8824 8548 Honors Thesis V 1 = 0.09 antibody (C-terminal) Data and Results Antibody Additions Stock Concentrations Desired Concentration Required Volume C terminal Hinge Region N terminal Well Number 1 2 3 4 5 6 Sample Table 1. The gel loading specifications Table 2 Primary antibody volume requirements
Dilan Patel 8824 8548 Honors Thesis Fig 5 C terminal antibody. Fig 6. Hinge region antibody. kDa kDa
Dilan Patel 8824 8548 Honors Thesis Fig 7. N terminal antibody.
Dilan Patel 8824 8548 Honors Thesis Conclusion Fig 8. O ptimal binding of CTGF
Dilan Patel 8824 8548 Honors Thesis
Dilan Patel 8824 8548 Honors Thesis References sv-SE
Dilan Patel 8824 8548 Honors Thesis
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