This protocol describes a method for removing antibodies reactive with bacterial-encoded proteins from polyclonal antisera by co-warming the antiserum with bacterial lysate. The absorption method described in this section is applicable only to the preparation of antisera containing low-titer anti-E. coli antibodies. This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
Cross-reactive antibody removal assay in antisera
Materials and Instruments
Closure buffer Closure solution Cell suspension buffer Antibody LB medium Move makings For more product details, please visit Aladdin Scientific website.
Sorval GSA rotor or equivalent Ultrasonic Breaker for handling bacteria E. coli Y1090 hsdR
Buffers and solutions
For components of storage buffers and reagents, see catalog 12.
Dilute the storage solution to the appropriate concentration.
Enclosure buffer
10 mmol/L Tris-Cl (pH 8.0)
150 mmol/L NaCl
0.05%(V/V)Tween-20
Sealing solution (1% m/V gelatin, 3% m/V bovine serum albumin, or 5% m/V skimmed milk powder)
The advantages of these containment reagents vary from laboratory to laboratory. It is recommended that the user perform a pre-test to determine which of these containment solutions is the best match for the primary and secondary antibodies of choice. The blocking solutions can be stored at 4°C and reused several times. Sodium azide at a final concentration of 0.05% (m/V) is added to inhibit microbial growth.
Cell Suspension Buffer
50 mmol/L Tris-Cl (pH 8.0)
10 mmol/L EDTA(pH8.0)
Filter to remove bacteria and store at 4°C in 50 ml aliquots.
Antibodies
Preparation of antibodies for library screening
Media
LB medium
Centrifuges and rotors
Sorval GSA rotor or equivalent rotor
Specialized equipment
Ultrasonic breakers for the treatment of bacteria
Carriers and strains
Escherichia coli Y1090 hsdR
This strain is available from Stratagene, Life Technologies or ATCC (www, atcc.org).
Methods
1. 100 ml of E. coli Y1090 hsdR strain was grown to saturation in LB medium.
2. Harvest the bacteria by centrifugation at 5000 g for 10 min at 4°C.
3. Resuspend bacteria in 3 ml of Bacterial Suspension Buffer. Repeat freeze-thawing several times, then ultrasonic treatment at 0°C on maximum power 6 times for 20 s each time.
4. The extract is centrifuged at 12000 g for 10 min at 4°C, the supernatant is transferred to another tube and the resulting lysate is stored at -20°C. The lysate is then stored at -20°C.
5. Before using the lysate, dilute the antibody preparation for screening 1:10 with the blocking solution.
6. Add 0.5 ml of E. coli lysate per ml of antiserum. Allow to incubate at room temperature with slow shaking for 4 h. Add 0.05% sodium azide to the processed antibody and store at 4°C for use in immunoscreening.