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| This protocol generates Keyhole Limpet Hemocyanin (KLH) carrier protein conjugates that can be coupled to a peptide of choice. This antigen is then used for immunization of rabbits for polyclonal antibody production. KLH, from the giant sea mollusk Megathura crenulata, is a very popular carrier protein because it is highly antigenic. This is due to its large mass and because it is a non-mammalian protein. |
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A. Checking Peptide Thiol Groups (see Hint #2)
1. Weigh out about 1 mg of peptide of interest into a tared 50 ml polypropylene conical tube (see Hint #3).
2. Add 0.5 ml of Ellman's Reagent. The solution should turn bright yellow.
3. Dilute the mixture 1:50 in 0.1 M Sodium Phosphate Buffer. Read the absorbance at the 412 nm (Abs412) wavelength, comparing the reading to the Abs412 of the reagent alone diluted to the same concentration.
4. Calculate the apparent molecular weight of the peptide based on thiol groups, using a molar extinction coefficient (εmax) of 14,000. Compare this to the expected molecular weight of the peptide. They should agree within a factor of three, with the estimated molecular weight usually higher (see Hint #4).
B. Coupling of Peptide to KLH
This protocol will yield enough peptide-KLH for the immunization of two rabbits with approximately five injections per rabbit (see Hint #5).
1.Weigh out 100 mg of Keyhole Limpet Hemocyanin (KLH) and dissolve the solid in 2 ml of ddH2O.
2. Sonicate and vortex to encourage the KLH to dissolve (takes approximately 4 hours), and mix the solution by continuous rotation using a "nutator" rotating platform or similar device at 4°C.
3. Once the KLH is dissolved, dialyze the KLH solution against 2 liters of 0.1 M Sodium Phosphate, pH 7.8, overnight at 4°C (see Protocol on Dialysis of Proteins). This removes any contaminating thiols or amino compounds.
4. Centrifuge 10 minutes at full speed in a microcentrifuge to remove aggregates (see Hint #6).
5. Split the dialyzed KLH solution into 2 aliquots: one for thiol- and one for amino- coupling.
C. Amino Coupling
1. For amino coupling, add 5 mg of peptide to one aliquot of KLH. Add the peptide as a solid if it is soluble in water; otherwise, add the peptide directly from a 100 mg/ml stock of peptide dissolved in DMSO. Precipitation does not matter and often happens.
2. Add Glutaraldehyde to a final concentration of 0.1% (CAUTION! see Hint #1).
3. After adding the Glutaraldehyde, check the pH of the peptide-KLH-Glutaraldehyde reaction by spotting 2 μl onto pH paper. If the pH is low, adjust to pH 7.8 using NaOH.
4. Incubate for 8 to 12 hr at 4°C, rotating gently on a nutator or equivalent.
5. Add a tiny pinch (a few milligrams) of Sodium Borohydride (NaBH4) to quench the remaining Glutaraldehyde. Make sure the sample is in a larger tube (i.e. 15 ml conical) since it tends to fizz up.
6. Incubate the sample for 8 to 12 hr at 4°C with gentle rotation. This is the "glut conjugate".
D. Thiol Coupling
1. For the thiol coupling, warm the second aliquot of KLH solution to room temperature.
2. Add 0.11 volume of IAA-NHS ester.
3. Incubate at room temperature. After 10 min, the KLH-IAA-NHS reaction will start to get a little cloudy.
4. Load the reaction onto a P-10 (Bio-Rad) column equilibrated with 0.1 M Sodium Phosphate, pH 7.8. Make sure the column is at least 10 times the volume of the sample.
5. Elute the KLH-IAA-NHS with 0.1 M Sodium Phosphate, pH 7.8.
6. Pool the KLH containing fractions by color (greyish green).
7. Add 5 mg of peptide to the KLH containing fractions. Add the peptide as a solid if it is soluble in water; otherwise add peptide directly from a 100 mg/ml stock of peptide dissolved in DMSO. Precipitation does not matter and often happens.
8. Add Glutaraldehyde to a final concentration of 0.1%.
9. After adding the Glutaraldehyde, check the pH of the peptide-KLH-glutaraldehyde reaction by spotting 2 μl onto pH paper. If the pH is low, adjust to pH 7.8 using NaOH.
10. Incubate for 8 to 12 hr at 4°C, rotating gently on a nutator or equivalent.
11. Add a tiny pinch (a few milligrams) of NaBH4 to quench the remaining Glutaraldehyde. Make sure the sample is in a larger tube (i.e. 15 ml conical) since it tends to fizz up.
12. Incubate the sample for 8 to 12 hr at 4°C with gentle rotation. This is the "glut conjugate".
13. Pool the coupled peptides (glut conjugates) from the amino and thiol couplings.
Dilute to 5 ml and add NaCl to 0.15 M final concentration. If there is a precipitate, sonicate vigorously to break it up.
14. Split the peptide-KLH into 1 ml aliquots (each aliquot will immunize two rabbits) and freeze them to store.
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| Ellman's Reagent |
| Prepare in 0.1 M Sodium Phosphate Buffer.
5 mM Dithio-bis-2-Nitrobenzoic acid
Make up fresh.
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| Iodoacetic Acid N-Hydroxysuccinimide (IAA-NHS) Ester |
| Protect from light.
100 mg/ml Iodoacetic Acid N-Hydroxysuccinimide Ester (Sigma)
Make up fresh.
Prepare in DMSO. (CAUTION! see Hint #1)
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| 5 M NaCl |
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| 0.1M Sodium Phosphate, pH 7.8 |
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| 0.1 M Sodium Phosphate Buffer |
| 0.1 M Sodium Phosphate, pH 7.2
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Dithio-bis-2-Nitrobenzoic acid
Iodoacetic Acid N-Hydroxysuccinimide Ester
DMSO
Sodium Borohydride
Sodium Hydroxide
Sodium Phosphate
Sodium Chloride
Keyhole Limpet Hemocyanin
Glutaraldehyde
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. Do this before thiol coupling, either to KLH or to resin. Peptide thiol groups have a tendency to get lost after synthesis.
3. Place the tube on a holder, place onto the scale of an analytical balance, and tare the balance.
4. If the thiol concentration is anomalously low (i.e. the apparent molecular weight is very high), there may be something wrong with the peptide and it will not couple well. You may need to regenerate the thiol groups by reducing the peptide with excess DTT and running a P2 column.
5. This procedure requires knowledge and training in the proper use and treatment of animals in experiments. Please consult with your institution's policies and instructions.
6. Don't be surprised to see a substantial pellet.
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