CHNG 2701: Fundamental of Bioprocess Final Report, 2004: Dr. Debashis Raha
Dot Blotting Group
Members:
Ch-ng Tau Lim
Poppy
Yang Li
Table of content
1.0 Work plan 3
1.1 MSDS 3
1.2 Theoretical Background 3
1.3 Procedures 4
1.4 Preparation of Solutions 4
1.5 Expected Results and Theoretical Conclusion 4
1.6 First And Second Experiment Run: 4
1.7 Results 5
1.8 Final Report with Discussion of Results 5
1.9 The final Presentation 5
2.0 Introduction 5
2.1 Choice of Membrane 6
2.2 Protein Properties 6
2.3 Antibodies Properties 7
2.4 Blocking Solution Used 8
2.5 Developing Solution Used 9
3.0 Procedure 9
3.1 Apparatus Required: 9
3.2 Solutions Needed in the Procedure 9
3.3 Preparations of Solutions 10
3.3.1 Preparation of Protein Solution 10
3.3.2 Preparation of Blocking Solution 11
3.3.3 Preparation of Antibody Solutions 11
3.3.4Preparation of Developing Solution 11
3.4 Run The Experiment 12
3.4.1 To Be Prepared Before The Experiment 12
3.4.2 Procedure 12
3.4.3 Diagram of The Procedure 14
3.4.4 Notes 14
4.0 Results 14
4.1 Discussion about The Result 15
4.2 Recommendations 16
5.0 Reference 17
1.0 Work Plan
The members of Group 3 have decided on dates and deadlines to complete the following laboratory assignment in this work plan. Our objective is to carry out a procedure and experimental technique and analyses to detect the presence of small quantities of particular protein. Besides, this work plan lets all the Group 3 members has an alert and guide about the group progression. Our plan will take into consideration of the following work outline:
Ø Collection of MSDS
Ø Theoretical Background
Ø Procedures
Ø Preparation of solutions
Ø Expected results and theoretic conclusion
Ø First experimental Run
Ø Second Experimental Run
Ø Result
Ø Final report with discussion of result
Ø Presentation to fellow students
Ø Laboratory demonstration
1.1 MSDS
The collection of all MSDS is already collected. This was carried out on the Monday Laboratory session 22nd of March. The MSDS were compiled from various company web sites from the World Wide Web. All the tems members should have a copied of these and to be aware of dangers and hazards of chemicals that are being handled during the experimental process.
1.2 Theoretical Background
This is the simply compilation and gathering of all our research data that are investigated to develop the practical procedures of dot blotting of proteins. This should be compiled and the final draft should be ready by the 3rd of April. Each member are expected to help in the compilation in the theoretical background. It is essential to clearly define our hypothesis and technique to be used as this is the main focal point of experiment.
1.3 Procedures
the procedure will be presented in two different methods:
1) In a form of flow-chart which will briefly outline the main experiment to be carried out.
2) A detailed procedure report, which will include preparation of regents and substance that need to be prepared before hand.
It will be prepared before 3rd of May
1.4 Preparation Of Solutions
The BSA solution shall be prepared few hours before the experiment. This is mainly why focused on the preparation of Tris Buffered Solution. This will be done the week before the actual first run. The preparation of this solution will be placed in charged by fellow member: Ch-ng Tau Lim
All the group members should make sure that all the required regents are available while undergoing the lab experiment. Besides, all the members should write a brief statement accounting for this, including making sure all other equipment is in working order. Before carry out procedure for the first run and for the preparation of the Tris Buffered solution, a permission to work forms should be signed by all the team members.
1.5 Expected Results and Theoretic Conclusion
The information for these sections will be compiled by Yang Li. This shall be prepared the next day after the preparation process. This is briefly a rough estimate of expected observation and conclusion. The purpose of it is to give a guideline of the expected result during first trail that will be carried out. Both of these sections will be treated as a guideline while comparing with the result of the initial run of the experiment.
These conclusions should be analysed by tutor to be certain of the group’s progress.
1.6 First And Second Experiment Run:
The first experiment run should be on 3rd of May and the final second run would be on 10th of May. For the second experiment, it should take into account of all the difficulties in experiment one. The group should prepare reports after each lab listing all technicalities. All readings and results should also be well documented in these reports.
1.7 Results
This will be the report prepared during the labs. This will be handed on 17Th of May and this will be compiled by Poppy, with the help of fellow members.
1.8 Final Report with Discussion of Results
At the final step, all relevant documents will be passed over to Ch-ng Tau Lim, who will prepare them for the final report. However, he will be given help from his fellow members with this tedious task when necessary. This report will be handed on 20th of May together with MSDS portfolio.
1.9 The final Presentation and Lab Demonstration
The final power point presentation slices will be compiled by Yang Li. All the group members will take part in the final presentation and lab demonstration on 20th of May.
This is the basic work plan of Group 3. All the group members should take this as a guideline to meet the each week tasks.
2.0 Introduction
Dot blotting was introduced in late 1970s to identify antigens that bound to specific antibodies. This is a method of applying proteins directly onto a membrane. Either applying a vacuum, absorption or intrusion pulls a dissolved sample through the membrane; protein binds to the membrane and the other sample components pass through. The proteins on the membrane are then available for analysis. This technique can be used either as a qualitative method for rapid screening of large number of samples or as a quantitative technique. It is especially useful for testing the suitability of experimental design parameters. However, we are using manual spotting methods for this experiment.
In this experiment, reagent-excess assay will be performed, during which the protein is bound to a membrane and probed with specific antibodies to form a detectable complex. The primary antibody attaches to the protein before a secondary antibody is attached to the original to form a complex. This complex acts as a catalyst in the reduction of hydrogen peroxide and a detectable coloured compound is formed in the presence of dye (4-chloro-1-naphthol). The procedure must be performed under sterile conditions to ensure the membrane is not contaminated with foreign proteins.
2.1 Choice of membrane
There are many choice of membrane, nitrocellulose, nylon membranes are all very good choices. Normally, nitrocellulose membranes are the most popular membranes. However, it is very fragile and has low binding capacity. Better membrane recommended would be nylon membranes, as they have six times binding capacity than nitrocellulose membranes. However, it is very expensive. So for this experiment, we chose nitrocellulose membrane.
Nitrocellulose membranes are the most popular membranes for immunoblotting (Western blotting) of proteins. The basis of protein binding to nitrocellulose is believed to be due to hydrophobic interactions. The 0.45µm pore size is the most commonly used for proteins greater than 10,000 MW, but for smaller proteins 0.2µm is recommended to more efficiently trap the proteins. The figure below is a guide of which kind of membrane should use for the experiment.
Figure 1: Guide to Protein Blotting Membranes
Due to the size of protein we used in this experiment, we use 0.45µm pore size membrane.
2.2 Protein Properties
Proteins are composed almost entirely of amino acids and hence have high molecular weights. Systematic classification of proteins is virtually impossible due to the number of different proteins in existence. Instead, they are classified according to biological function, source and occurrence. In this experiment, casein, ovalbumin, and gluten are used. The following are their properties.
Caseins belong to the phosphoroprotein group and are found in bovine milk (25g/L). They have three major groups: α-, β- and γ-caseins. γ-casein (MW 19093) is a single chain protein with 169 amino acids – 105 hydrophobic and 64 hydrophilic). There are six structurally similar α-caseins, containing approximately 200 amino acids each, and all except one are single chain molecules. β –casein (MW 24400) is 206 amino acids long.
Ovalbumin belongs to a group of pure proteins known as albumin and is found in chicken egg white. Albumins are easily crystallised, have a low molecular weight and are soluble in both water and salt solutions with pH between 4.0 and 8.5. Due to the high glutamic and aspartic acid content, they are also acidic. Ovalbumin contains 3.2 percent carbohydrates in one chain and a phosphoric acid reside attached to a serine side chain.
Found in wheat, gluten is made up of two proteins – glutenine and gliadin. Glutenine, containing about 45 percent glutamic acid, belongs to the gluteline group and is insoluble in water, salt solutions and ethanol. Glutenine is soluble in urea, reducing agents and dilute acids and bases. Gliadin, containing 30 to 40 percent glutamic acid and 15 percent proline, belongs to the prolamine group. It is insoluble in water and salt solutions, but soluble in 50-90% ethanol.
2.3 Antibodies Properties
As each antibody reacts to a specific antigen, they need to be chosen carefully so that an accurate indication can be made of which proteins are present. It is also of vital importance that the labelled antibody is chosen carefully to react with the first antibody.
In this experiment, Monoclonal Anti-Chicken Egg Albumin developed in rabbit is chosen as the primary antibody. The secondary antibody is an anti-rabbit IgG-peroxidase developed in goat, which reacts specifically with the rabbit IgG section of the primary antibody, forming the labelled final complex.
The antibodies used are immunoglobins, specifically IgG. This is a glycoprotein (MW 150 000) containing two identical heavy and two identical light polypeptide chains. The flexibility of immunoglobins is what facilitates their binding with antigens. The greatest flexibility is centred on the hinge region in the centre of the molecule. Two identical binding sites are composed from about 50 amino acid residues on the two branched ends of the molecule.
These binding sites bond with the antigenic determining regions of the protein, located at the lower end of the molecule. Around 20 amino acids from each reactant participate in this bonding. Each binding site (paratope) only bonds with a specific determining region (epitope), which is why IgG is so useful for performing immunoassays.
After the protein probing procedure has been performed, the conjugated peroxidase label can be detected. This is done by using hydrogen peroxide and a leuco dye (4-chloro-1-naphthol). The peroxidase molecule catalyses the reduction of hydrogen peroxide, oxidising the dye to form an insoluble coloured compound.
2.4 Blocking solution used
For meaningful results, the antibodies must bind only to the protein interest – not to the membrane. Non-specific binding of anti-bodies can be reduced by blocking the unoccupied membrane sites with an inert or non-ionic detergent.
Therefore, the blocking agent should meet the criterion below:
· Fill all unoccupied binding sites
· Not displace or bind to epitopes on the protein of interest;
· Have a greater affinity for the membrane than the antibodies; and
· Not cross-react with the chosen antibodies.
As BSA solution reaches the criterion above, it has been chose to use in this experiment.
2.5 Developing Solution used
4-Chloro-1-naphthol and hydrogen peroxide have been used as a developing solution in this experiment. It helps in the detection of active proteins. The amount of washing required is best determined experimentally. Too little washing will yield an excessive background, while too much may elute the antibody or protein and reduce the signal.
3.0 Procedure
The aim of this experiment is to confirm that the present of the ovalbumin among the proteins tested.
3.1 Apparatus Required:
· Balance (4 decimal places)
· Spatula of various size
· Volumetric flasks (250ml), (100ml), (500ml), (2L)
· Beakers of various size
· Pasteur pipette
· Measuring cylinder of various size
· Micro-pipetter of various size and tips
· MoCartney’s bottles
3.2 Solutions needed in the procedure
1) Bovine Serum Albumin (BSA)
2) Tris-Cl
3) Primary Antibodies
4) Secondary Antibodies
5) 4-Chloro –naphthol
6) 6% hydrogen peroxide
3.3 Preparation of solutions
Below are the methods and steps that used to prepare each of the solution.
3.3.1 Preparation of protein solution
1) Tris-Cl (Sigma, C/No: T-664)
Total amount needed—2.5L, 0.02M
Weight 2500*0.02=50g Tris-Cl solid into a clean and dry beaker, add distilled water to dissolve the entire solid, and then pool the liquid into a 2L volumetric flask, to make up a 2L solution. Using a measuring cylinder to divide the solution to 5*400ml solutions, and then transfer to a 5*500ml volumetric flask, to make up 5*500ml Tris-Cl solution
2) Urea (APS, C/No: 817-5kg, molecular weight: 60.06g/mol)
Total amount needed--250ml, 8M
Weight 0.25*8*60.06=120.12g urea solid into a clean and dry beaker, add distilled water to dissolve the entire solid, and then transfer to a 250ml volumetric flask
3) Gluten (Sigma, C/No: G-5004)
Total amount needed—10ml, 0.1 %(w/v) in urea
Weight 10*0.001=0.01g solid into a clean and dry MoCartney’s bottle. Add urea solution by Pasteur pipette into a measuring cylinder to obtain 10ml solution, and then transfer it into the MoCartney’s bottle, to make up approximately 10ml gluten solution
4) Casein (Sigma, C/No: C-7078, molecular weight 19,000-25,000g/mol)
Total amount needed—10ml, 0.1 %(w/v) in urea
Weight 10*0.001=0.01g solid into a clean and dry MoCartney’s bottle. Add urea solution by Pasteur pipette into a measuring cylinder to get 10ml urea solution, and then transfer it into the MoCartney’s bottle, to make up approximately10ml casein solution