| |
| This protocol is part of a procedure to affinity purify an antibody. In this protocol, a resin is activated and coupled to the peptide antigen. See the Protocol of Purification of Anti-Peptide Antibody to purify the antibody over this column. The contributor uses Affi-Gel 10, converting its functional group first to amino and then to iodoacetyl. You can also buy amino resin (it is more expensive) and start the protocol at Step #8. |
| |
1. Add 1 ml of Affi-Gel 10 resin (Bio-Rad) to a glass-fritted filter funnel that is attached to a vacuum line (save a 50 μl resin aliquot to verify resin preparation) (see Hint #2).
2. Wash the resin with 5 bed volumes of 100% cold Ethanol.
3. Wash the resin with 5 bed volumes of 50% cold Ethanol.
4. Wash the resin with 5 bed volumes of cold ddH2O.
5. Add 5% Ethylene Diamine to the resin.
6. Incubate at room temperature for 15 min.
7. Wash the resin with 10 bed volumes of ddH2O (save a 50 μl resin aliquot to verify resin preparation) (see Hint #3).
8. Wash the resin with 3 bed volumes of 0.1 M Sodium Pyrophosphate.
9. Resuspend the resin in 0.1 M Sodium Pyrophosphate.
10. Add IAA-NHS ester while gently stirring the resin with a glass stirring rod (see Hint #4).
11. Incubate at room temperature for 10 min (see Hint #5).
12. Wash the resin with 10 bed volumes of 0.1 M Sodium Pyrophosphate (save a 50 μl resin aliquot to verify resin preparation).
13. Resuspend the resin as a 50% slurry in 0.1 M Sodium Pyrophosphate.
a. Check the resin chemistry before wasting valuable protein as follows (see Hint #6):
b Resuspend each resin aliquot (saved in Step #1, #7, #12) in 100 μl of 0.1 M Sodium Pyrophosphate.
c. Add 1 μl of 0.1 M NHS-Fluoroscein or 0.1 M NHS-Rhodamine.
d. Incubate for 5 min at room temperature.
e. Centrifuge to pellet the resin, discard the supernatant and wash the resin twice in 0.1 M Sodium Pyrophosphate.
f. The original resin (Step #1), and the resin after Step #12 should be only lightly labeled. The resin after Step #7 should be heavily labeled.
14. If the peptide is soluble in 0.1 M Sodium Pyrophosphate add 5 mg peptide as a solid (see Hint #7).
15. Mix gently on a rotating wheel overnight at 4°C.
16. Add 2-Mercaptoethanol to 0.2% (v/v) (see Hint #8).
17. Incubate resin for 1 hr at room temperature.
18. Wash the resin with 5 bed volumes of 0.1 M NaHCO3.
19. Wash the resin with 5 bed volumes of 1 M Na2HCO3.
20 Wash the resin with 5 bed volumes of ddH2O.
21. Wash the resin with 5 bed volumes of 0.2 M Glycine.
22. Wash the resin with 5 bed volumes of 150 mM NaCl.
23. Wash the resin with 5 bed volumes of Guanidine Buffer.
24. Re-equilibrate the resin into Sodium Azide Buffer.
25. Continue with Protocol on Purification of Anti Peptide Antibody.
|
| Sodium Azide Buffer |
| 0.1% (w/v) Sodium Azide (CAUTION! see Hint #1)
Prepare in TBS Buffer
|
 |
| Guanidine Buffer |
| 6 M Guanidine-HCl
Prepare in TBS Buffer
|
|
| TBS Buffer |
| 20 mM Tris-HCl, pH 7.4
0.15 M NaCl
|
|
| 150 mM NaCl |
|
| 50% (v/v) Ethanol |
|
| 0.2 M Glycine, pH 2.0 |
|
| 0.1 M NHS-Rhodamine |
| Prepared in DMSO
Need either Fluoroscein or Rhodamine solution
|
|
| 1 M Sodium Carbonate (Na2HCO3) |
|
| 0.1 M NHS-Fluoroscein |
| Prepared in DMSO
Need either Fluoroscein or Rhodamine solution
|
|
| 0.1 M Sodium Bicarbonate (NaHCO3) |
|
| Iodoacetic Acid-N-Hydroxysuccinimide Ester (IAA-NHS) |
| Prepare in dry DMSO (CAUTION! see Hint #1)
Dissolve Iodoacetic Acid-NHS Ester (CAUTION! see Hint #1)
|
|
| 0.1 M Sodium Pyrophosphate, pH 7.8 |
|
| 5% (w/v) Ethylene Diamine |
| Prepare in ddH2O just before use
|
|
|
| |
2-Mercaptoethanol
NHS-Rhodamine
NHS-Fluoroscein
Iodoacetic acid-NHS ester (IAA-NHS)
Guanidine-HCl
Ethylene Diamine
Glass-fritted filter funnel
Affi-Gel 10
DMSO
Tris
Sodium Chloride
Sodium Carbonate
Sodium Pyrophosphate
Sodium Bicarbonate
Sodium Azide
Glycine
|
| |
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. All washes are performed on a glass-fritted filter funnel. Remove washes with vacuum until a wet cake is formed. Do not dry the resin completely or you will introduce air bubbles. All reactions up to peptide addition are performed in the funnel by covering the spout with Parafilm. This minimizes loss. Assume the resin is a 50% slurry.
3. At this point you have the amino-Affi-Gel .
4. The resin has approximately 10 μmol reactive groups/ml, therefore you can add approximately 20 μmol IAA-NHS reagent per ml of resin (7 mg IAA-NHS Ester/ml amino- Affi-Gel resin).
5. This step and subsequent steps up to the blocking of residual Iodoacetate groups should be done in dim light since the Iodo-group is light sensitive.
6. The amino resin will react with an NHS ester, whereas the original Affi-Gel and the iodoacetate will not. The procedure takes advantage of this chemistry to check whether the Affi-Gel is primed from protein addition.
7. Generally, if the peptide was readily soluble when it was checked for thiol groups, it will be soluble in the 50% slurry because the concentration is the same. Many peptides can be added as a 100 mg/ml stock in DMSO. Some hydrophobic peptides will crash out. It is possible to couple such peptides in 20% buffer, 80% DMSO. In an extreme case you can use 100% DMSO containing Triethylamine. The amount of peptide to add is difficult to predict and will require some fine tuning. The contributor of the protocol usually adds 1 to 2 mg peptide/ml resin, hoping to make a resin that will bind 10 to 100 mg/ml of specific antibody.
8. The 2-Mercaptoethanol will block residual Iodoacetate groups
|
