Selection of plasmid vectors to construct expression libraries
Selection of plasmid vectors to construct expression libraries
There are three methods for detecting bound antibodies: radiochemical screening, colorimetric screening, and chemiluminescent screening, the advantages and disadvantages of which are discussed in the introduction to this chapter. This experiment is from the next volume of the Molecular Cloning Laboratory Guide (3rd edition) by J. Sambrook D.W. Russell.
Operation method
Selection of plasmid vectors to construct expression libraries
Materials and Instruments
Blocking buffer Chloroform Lysis buffer Reagents for detection of antigen-antibody complexes SM TNT buffer Wash buffer 125I-labeled protein A or 125I-labeled immunoglobulin Radioiodinated secondary antibody Primary antibody LB or SOB agar plate Move makings For more product details, please visit Aladdin Scientific website.
Air incubators Flat-tipped forceps Syringes with 18-gauge needles filled with waterproof black ink (India ink) Cellulose nitrate filters Plastic cases and glass Petri dishes Whatman3 MM filter paper Plasmid expression libraries
Buffers and solutions
For components of stock solutions, buffers and reagents, see Appendix 1. dilute the stock solution to the appropriate concentration.
Seal the buffer:
10 mmol/L Tris-Cl (pH 8.0)
150 mmol/L NaCl
0.05% (V/V) Tween-20
20%(V/V) Fetal bovine blood淸
Other alternative containment solutions are 5%(m/V) skimmed milk powder or TNT buffer with 1%(m/V) gelatin and 3%(m/V) bovine serum albumin. The merits of the various reagents vary from laboratory to laboratory. It is recommended that the investigator perform pre-tests to determine which will work best in combination with the screening primary and secondary antibodies. The blocking buffer is stored at 4°C and can be reused several times. Sodium azide should be added at a final concentration of 0.05% (m/V) to inhibit microbial growth.
Chloroform
Lysis buffer:
100 mmol/LTris-Cl (pH 7,8}
150 mmol/LMgCl 2
1.5% (m/V) bovine serum albumin
1ug/ml Pancreatic DNAase I
40ug/ml Lysozyme
Reagents for detection of antigen-antibody complexes:
Reagents used for colorimetric screening using alkaline phosphatase (AP)-coupled antibodies
Reagents for colorimetric screening using horseradish peroxidase (HRP)-coupled antibodies.
Reagents for chemiluminescence screening
For the reagents used in each screening method, see step 19, and for common methods of antibody detection, see Immunoscreening with Antibodies in the information section at the end of this chapter in Appendix 9.
SM
Store at room temperature and discard after use to prevent contamination.
TNT Buffer:
10 mmol/LTris-Cl (pH 8.0)
150 mmol/LNaCl
0.05%(V/V)Tween-20
Approximately 1 liter of TNT buffer is required for each 10 membranes screened. Store at room temperature.
Wash buffer:
TNT buffer with 0.1% (m/V) bovine serum albumin.
TNT Buffer with 0.1% (m/V) bovine serum albumin and 0.1% (V/V) NP-40
These solutions do not contain sodium azide.
Radioactive Compounds
125I-labeled protein A or 125I-labeled immunoglobulin
Radioactive Iodide Secondary Antibody
This antibody can be used if an antigen-antibody complex is to be detected by the radiolabeled secondary antibody in the step. 
Antibody
Primary antibody
For the selection of primary and secondary antibodies, see the information section on Immunoscreening with Antibodies at the end of this chapter in Appendix 9.
Medium
LB or SOB agar plates
Contain antibiotics suitable for the construction of cDNA expression library systems or vectors. 30-35 ml of agar medium should be included in a 90 mm dish, or approximately 50 ml per 150 mm dish. the plate must be kept dry or the top layer of agarose will peel off when the nitrocellulose filter membrane is removed. Often it is better to open the lid slightly on a plate made 2 days ago and allow it to dry for 1-2 h at 37°C.
LB or SOB Agar Plates with 1 mmol/LIPTG
If the expression vector carries the lac promoter, IPTG-containing plates are required. For IPTG-induced protein expression, please refer to the discussion of expression vectors with IPTG-inducible promoters in the Introduction of Chapter 15.
Specialized Equipment
Air incubator at 30°C and 42°C
Required if the expression vector contains the λ phage PR promoter (e.g. pEX series); otherwise, the incubator can be set at 37°C.
Flat-tip tweezers
Syringe with waterproof black ink (India ink) and 18-gauge needle
Nitrocellulose filter membrane
Filter membranes suitable for binding proteins and immunoblotting reactions include Triton X-100-free nitrocellulose filter membranes (Millipore HATF or equivalent) and nitrocellulose derivatives such as Hybond-C extra (Amersham Pharmacia Biotech), whereas nylon membranes or nylon membranes with a polarity are not suitable for immunoscreening due to high background and weak ability to fix proteins. Nylon or polarized nylon membranes are not suitable for immunoscreening due to high background and weak protein immobilization.
Mark the membrane with a soft pencil or ballpoint pen. Wet with water and sandwich between dry Whatman 3 MM filter papers. Wrap stacks of filter papers in aluminum foil and autoclave at 10 psi (0.70 kg/cm2 ).
Plastic Boxes and Glass Petri Dishes
Stack of Whatman3 MM Filter Papers
Prepare one sheet of Whatman 3 MM filter paper per membrane with a slight excess. Wrap in aluminum foil and autoclave (0.70 kg/cm2).
Carrier and Bacterial Strain
Plasmid Expression Library
Construct cDNA libraries using suitable expression vectors as described in Chapter 11 or purchase from suppliers.
Additional Reagents
Reagents listed in Scheme 1 are required for Step 19.
Methods
Preparation of Main Plate and Membrane
1. Sterile flat-tip tweezers (e.g. Millipore tweezers) Place a sterilized nitrocellulose filter membrane on an LB (or SOB) plate, numbered side down. The membrane is then removed, inverted and repositioned with the numbered side facing up.
2. Take a small amount of bacterial solution (less than 0.5 ml per 138 mm membrane, which can contain up to 20,000 bacteria, or less than 0.2 ml per 82 mm membrane, which can contain up to 10,000 bacteria). Apply a sterilized elbow glass rod to the surface of the membrane, leaving a sterile border 2-3 mm wide at the periphery of the membrane. Leave the plate at room temperature until all liquid is absorbed.
3. Invert the plate and incubate (8-10 h) until very small colonies (0.1 mm in diameter) appear.
Colonies transformed with expression vector with lac promoter should be incubated at 37°C, while colonies transformed with expression vector with λ phage PR promoter should be incubated at 30°C to prevent expression of the fusion protein.
Preparation of Reproduction Filter Membrane
4. Wet a numbered and sterilized nitrocellulose filter membrane in contact with the surface of another agar plate containing the appropriate antibiotic, numbered side up, with the number of the duplicate membrane corresponding to the number of the primary membrane.
5. Using sterilized flat-tipped tweezers, gently transfer the primary membrane from the first plate (step 3) to a stack of Whatman 3 MM filter paper. The colonized side is facing up.
6. Carefully place the second wetted membrane (numbered side down) on top of the corresponding numbered primary membrane. Be careful not to move the membrane once it is in contact. Place a round piece of 3 MM filter paper on top of the membrane. Place an empty dish on top of the paper, with the bottoms touching, and flatten the dish with your hand to make a copy of the membrane.
7. When the two membranes are sandwiched together, use an 18-gauge needle to make a series of specifically marked positioning holes in the membrane. Gently peel off the membranes and place the duplicate membranes on a moistened used plate (step 4), and place the primary membrane on a fresh plate containing the appropriate antibiotic with the bacterial side up.
If required, several duplicate membranes can be prepared from a single primary membrane. However, if the primary membrane is to be used for the preparation of more than two duplicate membranes, it should be incubated for a number of hours to regenerate the colonies. In general, it is preferable to prepare only two duplicate membranes from a single primary membrane to avoid the problems associated with fuzzy colonies.
8. Repeat steps 4 to 7 until all primary membranes have been copied.
9. Induce gene expression in plasmids cloned with lac promoter as follows.
a. Warm the plates (master and duplicate) at 37°C until colonies of 1 to 2 mm in diameter appear. Generally colonies on the master plate reach the desired size very quickly.
b. Cool the master plate to room temperature, wrap it in plastic wrap and store it at 4°C until immunoscreening results are obtained.
C. Place a copy of the membrane, numbered side up, on a fresh plate containing lmmol/LIPTG pre-warmed to 37°C. Continue to incubate for 2 to 3 hours. Continue to incubate for 2~4 h
d. To induce expression synthesis of expression vectors with λ phage PR promoter (e.g., pEX vector, see Appendix 3), transfer the membrane onto a pre-warmed plate and incubate at 42°C for 2-4 h.
Handling of Filter Membranes for Colony Immunoscreening
10. In a chemical fume hood, remove the nitrocellulose filter membrane from the plate with flat-tipped tweezers and place on a moist paper towel. The filter membrane was covered with a plastic box, and an uncovered glass flat dish containing chloroform was placed in the plastic box as well. Bacterial colonies on the filter membrane were exposed to chloroform vapor for 15 min.
11. Place a small portion of the membrane in a dish containing lysate (6 ml per 82 mm membrane, 12 ml per 138 mm membrane). After all membranes have been submerged, the dish is placed on a rotating platform and the lysis buffer is slowly shaken. 12-16 h are required for colony lysis at room temperature.
12. Transfer the membranes to TNT-containing Petri dishes or glass trays and incubate for 30 min at room temperature.
13. Replace with fresh TNT buffer and repeat step 12.
14. Place the membranes one by one in a glass tray containing TNT buffer and remove any colonial residue from the surface of the membrane with Kimwipes.
Detection of Expressed Fusion Protein Clones
IMPORTANT: Do not allow the membranes to dry out during the following steps. Antibodies that would otherwise bind non-specifically and reversibly to the moist membranes will remain permanently on the membranes once they have dried out, and it is important to prevent the membranes from becoming attached to each other when they are immersed in the various buffer solutions and antibody solutions. Place the dishes on top of each other on a low-speed platform shaker.
15. After all membranes have been stripped and rinsed, place them one by one in freshly changed TNT buffer. After all membranes have been transferred, continue to gently agitate the buffer for 30 minutes at room temperature.
If necessary, the membranes can be removed from the buffer at this point, wrapped in Saran wrap and stored at 4°C for 24 hours.
16. Using flat-tipped forceps, transfer the membranes one by one to a glass dish containing containment buffer (7.5 ml per 82 mm membrane, 15 ml per 138 mm membrane), submerge all membranes, and slowly shake the solution on a rotating platform for 30 min at room temperature.
17. Using flat-tipped forceps, transfer the membranes to fresh glass dishes containing diluted primary antibody in blocking buffer (7.5 ml per 82 mm membrane, 15 ml per 138 mm membrane), using the highest antibody dilution that produces a background that is not too high but still detects 50-100 pg of denatured antigen. All membranes are submerged and shaken slowly on a rotating platform for 30 min at room temperature.
The antibody can be stored at 4°C and reused several times. Microbial growth is inhibited by the addition of sodium azide to the solution at a final concentration of 0.05% (V/V).
18. Wash the membranes for 10 min in each of the following buffers, transferring the membranes sheet by sheet between buffers, using 7.5 ml of buffer for each 82 mm membrane; 15 ml for each 138 mm membrane.
TNT Buffer with 0.1% Bovine Serum Albumin
TNT Buffer with 0.1% Bovine Serum Albumin and 0.1% NP-40 (Nondidet P-40)
TNT Buffer with 0.1% Bovine Serum Albumin
19. Detection of antigen-antibody complexes by radiochemical, colorimetric or chemiluminescent reagents of choice.
Radiochemical screening
Approximately 1uCi125I-labeled protein A or immunoglobulin is required per membrane, and radiolabeled protein A is commercially available (specific activity 2-100uCi/ug). Radioiodine-labeled secondary antibodies can be purchased from the supplier or can be prepared as described in the information section of the material related to radioiodine labeling of IgG.
a. Dilute the radiolabeled ligand with containment buffer (7.5 ml per 82 mm membrane, 15 ml per 138 mm membrane).
b. Allow to incubate for 1 hour at room temperature, rinse several times with TNT buffer, and perform autoradiography as described in Appendix 9.
Continue to Step 20.
Color Reaction Screening
Antibodies coupled to horseradish peroxidase (HRP) or alkaline phosphatase (AP) that recognize the species-specific determinant of the first antibody can be purchased from the manufacturer and used at the dilutions recommended and required by the product insert. Typically, 5ul of coupled antiserum can be added to 7.5ul of blocking buffer (without sodium azide) per 82 mm membrane. For more information on HRP or AP, see Appendix 9.
Localization of Antigen-Antibody-Antibody-AP Complexes by Combined Application of 5-Bromo-4-Chloro-3-Indole Phosphate (BCIP) Substrate and Nitroblue Tetrazolium (NBT) 
a. Place the membrane in a solution containing the AP-coupled antibody and shake slowly for 1.5-2 h at room temperature. b. Place the membrane in a solution containing the AP-coupled antibody and shake slowly for 1.5-2 h at room temperature.
b. Wash the membrane according to step 14.
c. Prepare BCIP (50 mg/ml in 100% DMT) and NBT (50 mg/ml in 70% DMT) storage solution and store away from light.
d. Prepare BCIP/NBT development solution before use:
I. Add 33ul of NBT solution to AP buffer and mix well.
II. Add 16.5ul of BCIP storage solution, mix well, keep away from light, and use within 1h.
e. Blot the membrane with paper towel.
f. Immerse the membrane in BCIP/NBT Developer (7.5 ml per 82 mm membrane, 15 ml per 138 mm membrane) and incubate at room temperature for several hours.
g. Wash twice with distilled water to achieve a dark purple coloration at the antigen-antibody complex.
Continue to step 20.
Localization of antigen-antibody-antibody-HRP complexes using 4-chloro-1-naphthol 
a. Place the membrane in a solution containing the HRP-coupled antibody and shake gently for 1.5 to 2 h at room temperature.
b. Wash the membrane according to step 14.
C. Dissolve 60 mg of 4-chloro-1-naphthol in 20 ml of ice-cooled methanol to make developer solution. Prior to use, dissolve 60 mg of 4-chloro-1-naphthol in 100 ml of methanol.
Before use, mix with 100 ml of 10 mmol/L Tris-Cl (pH 7.5), 55O mmol/L NaCl solution containing 60 ul30% H2O2.
d. Blot the water from the membrane with a paper towel, and wash the membrane with 10 mmol/LTris-Cl (pH 7.5), 150 mmol/LNaCl solution.
e. Immerse the membranes in 4-chloro-1-naphthol developer (10 ml per 82 mm membrane, 10 ml per 138 mm membrane).
(10 ml for each 82 mm membrane, 25 ml for each 138 mm membrane) and incubate at room temperature for 15~20 min.
f. Wash the membrane twice with distilled water, and then the antigen-antibody complexes will show dark purple color.
Biotinylated species-specific antibodies and anti-biotin protein-coupled HRP are also commercially available and should be diluted according to the instructions of the different manufacturers and immunoscreened as described above for HRP-coupled antibodies.
Continue to step 20.
Luminescence Screening
The chemiluminescent reaction is the most sensitive method for detecting immunopositive phage. Secondary antibodies are generally coupled to AP or HRP, and substrates such as 1,2-dioxetane phosphates are required when using AP-coupled antibodies. This phosphorylated substrate emits light at 466nm, and the HRP-coupled antibody oxidizes the substrate luminal, which emits a strong light at 428nm in the presence of hydrogen peroxide and phenol. In both systems, the light emitted can be captured by autoradiography. Chemiluminescent detection is rapid, sensitive (1-10 Pg of antigen can be detected), and results in a permanent record of the screened film (x-ray film). Potential disadvantages are the high cost of reagents and the need to compare the autoradiography film with a master plate to obtain a positive clone, as shown in the typical protocol below. 
a. Place the filter membrane in a solution containing an AP or HRP coupled antibody and shake gently for 1.5 to 2 h at room temperature.
b. Wash the membrane according to step 18.
c. Prepare the chemiluminescent reaction substrate according to the manufacturer's instructions.
d. Incubate the washed membrane with the chemiluminescent substrate for 1 to 5 min (determine the optimal exposure time according to the manufacturer's recommendations).
e. Remove excess liquid from the filter membrane and wrap immediately in Satan wrap, do not allow the membrane to dry.
For radiographic autoradiography (see Appendix 9), the initial exposure is usually 1 min, and the appropriate exposure time is determined at this interval.
Continue to step 20.
20. Identify the location of the positive clone or compare to a duplicate filter membrane for a consistent signal. For radiolabeled or chemiluminescent probes, compare the agar plate in the light box with the results of the radioautography. For color-generating reagents that will leave a visible positive residue on the filter membrane, continue with the following steps.
a. Place a stack of Saran Wrap or Mylar membranes on the filter membrane.
b. Mark the location of the wells on the membrane and the location of the antigen-positive clones on the surface of the Saran Wrap with a different color waterproof marker. the Saran Wrap should also be marked so that the plate corresponding to the membrane can be found.
c. Place the membrane on the surface of the reading box and check the plate containing the original colony by placing it on the membrane.
d. Determine the area of positive phage spots and transfer the agar block containing this area of presumed positive clones to 1 ml of LB culture medium containing the appropriate antibiotic. Incubate for 12-16 h at appropriate temperature.
e. Save the Saran packaging membrane as a permanent record of the localization of positive phage spots. The discoloration on the original filter membrane will fade quickly.
21. Repeat the spreading and screening process until consistent immunopositive clones are obtained.
22. Identify immunoscreened isolated clones according to the method provided in Determination of Immunoscreened Isolated Clones in the Information column at the end of Scheme 1.