For N-terminus sequencing, Trypsin digest of NC membrane-bound proteins, elute, and collect peptide bands through HPLC (page 10-136).

For MS identification,

In-gel trypsin digestion, and run HPLC.

Immunoprecipitation (IP) with recombinant proteins

1. Sample, protein A or protein G-agarose beads, and normal mouse IgG; 40C for 40 min on an orbital shaker.

2. Centrifuge3. Supernatant, epitope-specific Ab, and protein A or protein

G-agarose beads; 40C for 3-4 hr on an orbital shaker.4. Centrifuge5. Wash pellet, and add SDS-sample buffer6. Boil, centrifuge, 7. Analyze supernatant on SDS-PAGE.

Immunoprecipitation can also be done with antibody-sepharose, or with 10 and 20 antibodies (20 antibo

dies recognizing F(ab)’ )(Fig. 10.15.2).

Immunoprecipitation-recapture

For more specific detection, or analyze the subunit components of a protein complex.

1. Add elution buffer containing 1% SDS to beads with bound antigen, votex

2. rt for 5 min, 950C for 5 min, cool to rt.3. Add BSA and mix4. Add lysis buffer containing 1% Triton 100, rt for 1

0 min5. Clear the lysate, and perform second immunopr

ecipitation (recognizing denatured protein).

Synthesized proteins of cloned genes in vitro

1. Sub-clone the coding sequence to a plasmid that contains a promoter for SP6, T7, or pSP64 promoter at a site downstream of a promoter.

The protein-coding sequences must be contiguous (no intron), the first AUG is 25 base to 100 base to 5’ end of RNA.

2. Prepare a good-quality plasmid DNA.3. RE cut at 50 to 200 base 5’ to the termination codon 4. Set up in vitro transcription reaction (polymerase, NTP, RNa

sin).5. Phenol and ethanol precipitation6. Add in vitro translation kit (wheat germ extract, reticulocyte l

ysate, or E. coli extract)7. 35S methionine is needed for radiolabelling. 8. SDS-PAGE.

Metabolic labeling of cells with radio-labelled amino acids.

To study biosynthesis, processing, intracellular transport, secretion, degradation, and physicoche

mical properties of proteins

Pulse - labelling Pulse - chase labelling

Long term labeling

For detection of T7 promoter-driven proteins, Add rifampicin to inhibit E. coli RNA polymerase

before labelling.

(Chen, et al., 2003, FEMS Microbol. Letts 224: 277-283)

Chapter 11: Immunology

Conjugation of or alkaline phosphatase (AP) (or horhorseradish peroxidase (HRP)) to antibodies

1. Dialyze antibody solution against PBS.2. Add AP and glutaraldehyde, incubate at rt. 3. At 5, 10, 15, 30, 60, 120 min, take aliquots to find

the best conjugation time by using a direct ELISA.

4. Dialyze the conjugate against PBS.

Indirect ELISA To detect specific antibodies: antibody screening, epitope mapp epitope mapping.- Useful when milligrams of purified or semi-purified antigen are available (1 mg antigen for screening of 80-800 microtiter plates).

1. Determine the optimal conc. of antigen solution and developer by criss-cross analysis.

2. Add antigen to microtiter plates, rt for overnight or 370C for 2 hrs. Wash.

(Pure antigen is not essential, >3% of total proteins need to be antigen, protein conc. is < 10 μg/ml.)

3. Add blocking (with BSA or geletin), rt for 30 min. Wash. 4. Add dilutions of antibody, rt for > 2hrs. Wash. 5. Add blocking, rt for 10 min. Wash. 6. Add developer, develop and read. protein A-AP (sigma), or anti-Ig AP conjugate (20 Ab). Substrate: NPP, chromogenic, use 405 μm filter. MUP, fluorogenic, use 365μm excitation filter, and 450μm emission filter.

Criss-cross analysis

1. Prepare dilutions of coating reagent (eg. 10, 5, 2.5, and 1.25 μg/ml), add to microtiter plates. Incubate and wash.

2. Prepare dilutions of second reagent (eg. 200, 50, 12.5, 3.125, and 0.78 μg/ml), add to microtiter plates. Incubate and wash.

3. Prepare dilutions of developer (eg. 200, 50, 12.5, 3.125, and 0.78 μg/ml), add to microtiter plates. Develop and read.

4. Find the conc. of coating reagent, second reagent, and developer to get 0.50 absorbance /hr at 405 nm when use NPP as a substrate, or 1000 to 2000 fluorescence units /hy when using MUP as a substrate.

Direct competitive ELISA

To detect soluble antigens: antigen screening. - Useful when both a specific antibody and milligrams of purified or semi-purified antigen are available.

1. Determine the optimal conc. of coating antigen and conjugate by criss-cross analysis.

2. Coat antigen to microtiter plates, block, and wash.3. Prepare serial 1:3 dilutions of standard antigen in blocking b

uffer. 4. In a round-or cone-bottom microtiter plate, add conjugate (s

pecific antibody-AP conjugate), followed by standard antigen, test antigen, and blocking buffer, rt for > 30 min.

5. Transfer 4. to 2. (above), incubate, Wash.6. Develop and read.7. Prepare standard curve (linear). X axis: plot standard antigen conc. on a log scale.

Y axis: absorbance or fluorescence.

Antibody-sandwich ELISATo detect soluble antigens: antigen screening. - 2-5 fold more sensitive than direct competitive ELISA. - Need large amounts of purified or semi-purified specific a

ntibody (capture antibody), antigen, and purified or semi-purified specific antibody recognizing epitopes distinct from those recognized by capture antibody. `

1. Determine the optimal conc. of capture antibody and conjugate by criss-cross analysis

2. Coat microtiter plates with specific antibody (capture antibody),

3. Block and wash.4. Prepare serial 1:3 dilutions of standard antigen. 5. Add standard antigen, and test antigen. Incubate and wash.6. Add developer (specific antibody-AP conjugate), develop a

nd read.7. Prepare standard curve (linear). X- axis: plot standard antigen conc. on a log scale. Y axis: absorbance or fluorescence.

(For Direct competitive ELISA & Antibody-sandwich ELISA)

Preparation of bacterial cell lysate antigens

1. Take 5 ml overnight cultures or 10 colonies, in 10 mM HEPES2. Add lysozyme solution, 5 min on ice.3. Centrifuge, save supernatant, 4. Resuspend pellet in TEN buffer, and add 10% SDS or 8M ure

a to solubilize proteins. 5. Analyze both supernatant from 3. and pellet proteins from 4. b

y ELISA).

Double antibody-sandwich ELISA

To detect specific antibodies: antibody screening, epitope mapping.

- Need small amounts of specific antibody and antigen. - But does not require purified antigen. - Can also be used for epitope mapping of different monoclon

al antibodies.

1. Coat microtiter plates with anti-Ig antibody not recognizing antigen or conjugate (capture antibody).

2. Block and wash.3. Prepare dilutions of test antibody. Add to microtiter plates, i

ncubate and wash.4. Prepare dilutions of antigen. Add to microtiter plates, incubat

e and wash.5. Add developer (specific antibody-AP conjugate, not recogn

izing capture antibody), develop and read. 6. For each positive, check again with blocking buffer replacing

antigen.

Direct cellular ELISA

To detect cell-surface antigens: antigen screening, surface antigen measurement.

- Sensitive for bulk screening - Need specific-AP conjugate.

1. Determine the optimal conc. of cells per well and conjugate by criss-cross analysis.

2. Add cells to round-or cone-bottom microtiter plate (centrifuge, aspirate supernatant, and votex to disrupt pel

let)3. Add conjugate, develop, and read.

Indirect cellular ELISATo detect antibodies specific for surface antigens: antibody s

creening. - Need positive control antibody (react with experimental

cells), negative control antibody (do not react with experimental cells).

1. Determine the optimal conc. of cells per well and conjugate

by criss-cross analysis.2. Add cells to round-or cone-bottom microtiter plate. 3. Add positive control, negative control, and test antibody, in

cubate and wash.4. Add anti-Ig-AP conjugate (20 Ab), develop and read.

Immuization of mice

Can use soluble antigen, acrylamide gel containing antigen, membranes, whole cells, micro

organisms for immunization.

Production of monoclonal antibodies- Purification of monoclonal antibodies using protein A- sepharose.

Production of polyclonal antibodies- Purification of polyclonal antibodies with saturated ammonium sulfate - Purification of polyclonal antibodies with DEAE-Affi-Gel Blue (Bio-rad).

Immunization with synthesized peptide

I. Selection a peptide, a) Choose 10- to 15 residue peptide corresponding to the N-

terminus and C-terminus of an antigen, should not be hydrophobic, basic residues are not good,

eg. C-terminus sequence SYGRNQAEKQ will be modified to CSYGRNQAEKQ

N-terminus sequence, similar, adding C residue at C-end for X-linking by MBS.

(C is a linker for X-linking by MBS).

MBS: m-Maleimidobenzoyl-N-hydroxysuccinimide ester.

Acetylation might be needed for some N-blocked antigen.

b) an internal sequence, based from algorithms for 1) Surface exposed, i.e., hydrophilic region, within a hydrophobic region, 2) conformationally flexible, e.g., a loop region, or a β-turn region. Avoid the same regions of the self-proteins in the immunized species.

2. Cross-link the chemically-synthesized peptide to a carrier proteins at C-terminus or N-terminus by MBS or glutaraldehyde.

- Carrier protein: KLH (keyhole limpet hemocyanin, very immunogenic) BSA (bovine serum albumin, very soluble)

- MBS: X-link thiol-group in C residue to lysine residue in carrier protein.

- Glutaraldehyde: X-link amino-groups of peptide and carrier. Peptide having lysine at positions other than N-terminus ar

e avoided.

3. Protein fractionation to obtain only the X-linked antigen may be required.