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how-to-prepare-peptide-protein-conjugates-for-immunization

How-to-prepare-peptide-protein-conjugates-for-immunization

 

[A] Check for the presence of thiol groups in the peptide.

Before the conjugation reaction, ensure that the peptide has a free thiol group needed for conjugation to keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or a resin. Peptide thiol groups tend to oxidize after synthesis.

  1. Prepare a 5 mM Ellman's reagent (dithio-bis-2-nitrobenzoic acid) in 0.1M sodium phsophate (NaPi) pH 7.2.

  2. Weigh out approximately 1 mg of the peptide into a reaction vial.

  3. Add 0.5 ml reagent. It should turn bright yellow.

  4. Dilute the mixture 1/50 in the buffer. Read A412 against reagent at the same concentration.

  5. Based on thiol groups, calculate the apparent molecular weight of the peptide using a molar extinction coefficient of 14,000. Compare this to the expected molecular weight of the peptide. The number should agree within a factor of three, with the apparent molecular weight usually being higher. Suppose the thiol concentration is very low and the apparent molecular weight is very high. There may be something wrong with the peptide, and it probably will not couple well—the thiol groups will need to be regenerated by reducing the peptide with excess DTT and desalted using a P2 column.

[B] Coupling of Peptide to KLH

This protocol produces enough conjugated peptide used for two rabbits using five injections per rabbit

  1. Weigh out 100 mg of keyhole limpet hemocyanin (KLH). Dissolve in 2 ml water. It generally takes about 4 hours to dissolve. Sonicate and vortex the solution. Be patient and put on a rotator at 4 deg C. Dialyze against 2 l of 0.1M sodium phosphate pH 7.8 overnight to remove any contaminating thiols or amino compounds.

  2. Spin 10 minutes at full speed in microfuge to remove aggregates (You may observe a substantial pellet).

  3. Split the KLH into two (2) aliquots for -SH and -NH2 coupling.

  4. For -NH2 coupling, add 5 mg peptide to one aliquot, followed by glutaraldehyde to 0.1% final. Add the peptide as a solid if it is soluble. Otherwise, use a 100 mg/ml stock in DMSO. Precipitation does not matter and often happens. After adding the glutaraldehyde, check the pH with pH paper, and adjust to 7.8 if necessary using NaOH. Incubate 8-12 hrs at 4 degrees, rotating gently.

  5. Add a tiny pinch of NaBH4 to remove the remaining glutaraldehyde. Ensure the sample is in a large tube since it tends to fizz up. Incubate 8-12 hrs at 4 degrees. The result is the glutaraldehyde conjugate.

  6. For the -SH coupling, warm the other aliquot of KLH to room temp. Add 1/9th volume of Iodoacetic acid N-hydroxysuccinimide ester at 100mg/ml in DMSO. Make the DMSO stock fresh, and protect the iodoacetamide reagent from light. You can make your own IAA-NHS ester, but it is available from Sigma.

  7. After 10 minutes at room temperature, the KLH will get a little cloudy. Load it onto a P-10 column equilibrated with 0.1M sodium phosphate at pH 7.8. Make sure the size of the column is at least ten times the volume of the sample. Pool the KLH containing fractions by color (it will look greyish green). Add 5mg of the peptide to the solution, as in step 5 above. Incubate at least 8 hrs at 4 degrees, rotating gently.

  8. Pool the coupled peptide from the two procedures. Dilute to 5ml with 0.15M NaCl. If there is a precipitate, sonicate vigorously to break it up. Split the immunogen into 1 ml aliquots (each aliquot will immunize two rabbits) and freeze it.