Purifying+antibodies+using+random+peptides+on+TentaGel+Beads+in+Column+Protocol

(Protocol adapted from Current Protocols in Immunology 9.3 Basic Protocol 3)
 * Purifying antibodies using random peptides on TentaGel Beads in Column Protocol**

TentaGel beads with synthesized peptide Column Equilibration buffer Antiserum known to contain anti-peptide antibodies Neutralization buffer Elution buffer (choose one of the following): PBST 0.45-μm syringe filter with 3- or 5-ml syringe with Luer lock (needed if you plan to "clarify", but this may not be necessary) Slide-A-Lyzer for dialysis 100, 70, 50, and 30% DMF Protein-lo bind tubes (cat no 022431081 from Eppendorf (can be ordered through VWR or many others))
 * Materials**
 * Note that an aliquot can be taken to Life Sciences for N Terminal sequencing to verify the identity of the peptide if desired. John Lopez runs the sequencer in the Protein core he will need very little material 2-3 beads. It will cost $5 per residue.
 * Zhao mentinoed that it may be better to use Sephadex beads rather than Tentagel beads for antibody purification
 * disposable 5 mL polypropylene columns (prod # 29922 Thermo)
 * 5 mL plastic columns at cabinet at left side of scanner room door
 * current protocols suggests: 50 mM potassium phosphate (pH 7.5)/0.4 M NaCl with and without azide
 * Bart and Kaitlyn have used TBST. Bart mentioned that I'll have higher background without TBST.
 * Shen uses 21001 Protein A IgG Binding Buffer
 * current protocols suggests: 1 M Tris⋅Cl, pH 7.5
 * Bart and Kaitlyn have used 1.5 M Tris 150 mM NaCl pH8 before
 * Tris MW 121.14 g/mol
 * Tris: 1.5 mol/L*121.14g/mol*1L = 181.71 g Tris
 * NaCl: 150 mmol/L*1*mol/(1000*mmol)*58.5*g/(1*mol)*1*L= 8.78 g NaCl
 * about 50 mL of 6 N HCl was needed to reach pH 8
 * Acidic elution buffer: //0.1 M glycine (pH 2.8)/0.15 M NaCl//, 0.1 M acetic acid, or 0.1 M sodium citrate, pH 3.0 (see recipes)
 * Bart has used 0.1 M citric acid pH 2.3
 * glycine: 0.1*mol/L*75.07*g/mol*1*L = 7.507 g glycine
 * 150 mM NaCl -> 8.78 g NaCl
 * about 5 mL 6 N HCl required to reach pH 2.8
 * Basic elution buffer: 0.1 M diethanolamine, pH 9.8 (see recipe)
 * Chaotropic elution buffer: 4 M sodium isothiocyanate, pH 7.5, 6 M urea, pH 7.5,
 * 5 M guanidine⋅HCl, pH 7.5 (see recipes)
 * Shen uses 21004 IgG Elution Buffer
 * Don't use polystyrene or polycarbonate with DMF. You can use glass and polypropylene with DMF. Most pipette tips are polypropylene. Generally plastic that is completely clear cannot be used with DMF, but if it is slightly opaque then it is often okay.

>> 1-cm-diameter column, keep the flow rate for all steps below 1 ml/min. Approximately 1 >> drop per second is good. >> Antibodies absorb well in the near UV region, so monitoring anywhere in the range of 254 >> to 280 nm is acceptable. The baseline should level off at <0.1 AU. >> 0.45-μm syringe filter attached to a 3- or 5-ml syringe with Leur lock. >> of in a column. In this case, combine the sample with 3 to 4 ml resin in a tube and gently >> tumble overnight at 4°C. Pour the resin into the column and continue with step 6. This >> alternative allows unattended extended sample loading for convenience. >> Typically, the antiserum contains a high concentration of anti-peptide antibodies; thus, >> antiserum that flows through the column still contains a significant amount of useful >> antibodies. For economy, this flow-through may be saved and rechromatographed. >>> method. >> fractions directly into the tubes containing neutralization buffer. >>> gently to mix them thoroughly with the dense neutralization buffer in the tube. >> changes of 100-fold excess volume at 4°C PBST. Aliquot as desired and store the >> antibody solution at −70°C. >>> minimal antibody. >> or without azide, depending on if the column is to be used again immediately or stored >> at 4°C. >>> microbial contamination, store the column refrigerated in equilibration buffer with >>> azide. Depending on the sample, and elution and storage conditions, expect 3 to 20 useful >>> runs, perhaps more.
 * Procedure**
 * If the peptide has just been synthesized onto the beads, gradually equilibrate the beads from an organic solution to an aqueous solution
 * May want to make a 20% bead slurry in DMF.
 * May want to use 100 uL of beads
 * Prepare 100, 70, 50, 30 % DMF (10 mL each) if these solutions are not prepared already
 * DMF last seen in chemical hood (B225 A)
 * DMF reacts with polystyrene transfer pipettes so use glass if possible. I used a glass aspirator pipette from box next to pH meter connected to a bulb
 * I think polycarbonate is okay
 * Any waste DMF should go in organic waste
 * Dissolve beads with peptide in organic solvent such as DMF and gradually move them into an aqueous solution
 * Add 10 mL DMF to column supported by equipment to hold the tube up and let the liquid drain off into a waste container
 * Add 10 mL 70% DMF. Wait for it to drain. Then add 50% DMF. Wait. Then add 30% DMF. Wait
 * draining takes some time
 * Wash beads with 5 mL PBS
 * Inspect the affinity column. If it shows signs of microbial growth, discard it. If it has a few air bubbles, unpack and repack it.
 * Pre-equilibrate the column by washing with equilibration buffer (such as TBST) until the monitored absorbance (i.e., fractions or online UV monitor) reaches a baseline. Alternatively, it is probably fine to flow a set volume through such as 5 mL. Note that if the column was just washed with DMF and then PBS this step is probably not necessary.
 * The column may be run either under gravity or with a pump. For best results with a
 * Prepare the sample by adding 1 to 2 ml antiserum known to contain anti-peptide antibodies to an equal volume equilibration buffer (such as TBST). Another dilution might be reasonable as well. Bart recommends a 1 to 10 or 1 to 100 dilution (1 to 100 dilution may be too small for many applications).
 * While dilution is not absolutely necessary for effective use of this protocol, it decreases viscosity and improves flow.
 * Clarify diluted serum sample
 * For clarification using a syringe filter: Pass the diluted serum sample through a
 * Removing particulate material from the sample improves flow and prolongs column life.
 * For clarification by centrifugation: Centrifuge 10 minutes at 1000 × g, room temperature.
 * Apply the sample to the column. Collect the antiserum that flows through unbound. Note that it may be a good idea to incubate the sample with the column on the rotisserie for 1 to 2 hr with the column capped and with parafilm on the bottom. Alternatively an incubation on the rotissery at 4 C overnight may result in even more cognate specific binding. Later on remove the top cap, and then the bottom parafilm
 * An alternative is to load the sample onto the pre-equilibrated affinity resin in batch, instead
 * May want to collect unbound antibody for storage since there could be some valuable antibodies in this solution.
 * Wash the column
 * current protocols suggestion: Wash the column with equilibration buffer (such as TBST), monitoring the eluent until UVabsorbance returns to baseline.
 * Use at least 20 ml potassium equilibration buffer (such as TBST) for this step.
 * Prepare for elution collection
 * current protocols suggestion: Load ten test tubes (best to use protein-lo bind tubes to prevent the small amount of antibody present from sticking to the sides of the tube) with 0.2 ml neutralization buffer if acidic or basic elution is to be performed, or water if chaotropic elution is to be used.
 * See Troubleshooting, Problematic Affinity Chromatography for a discussion of elution
 * Elute bound antibody
 * current protocols suggestion: For acidic elution: Elute the column with 5 ml acidic elution buffer. Collect 1-ml
 * Although not usually necessary, as soon as they are eluted, the antibodies may be swirled
 * Nanodrop the elution fractions to determine which ones contain antibody
 * Pool fractions containing antibody
 * May want to save some non-dialyzed sample
 * Perform dialysis on collected antibody solution
 * current protocols suggestion: dialyze (APPENDIX 3H) against at least two
 * Usually only three to five fractions will be pooled, since the flanking fractions contain
 * use Slide-A-Lyzer to perform dialysis
 * Re-equilibrate the column
 * current protocols suggestion: by washing it with 20 ml equilibration buffer (such as TBST) with
 * To prolong column life, re-equilibrate it as soon as possible after elution. To prevent

Questions about protocol What type of column will I use? plastic 5 mL column

How many Tentagel beads will I add to the column? if you have hyperimmune sera, use all of them (~0.5 mL) if you do not have hyperimmune sera or low titer sera use less maybe 100 ul but the problem is smaller volumes mean higher flow rate through the column so you would have to batch bind/batch elute.

What if I want to use less than 1 to 2 mL serum? Is this okay? Bart: You can use any volume you want. You would just use a smaller volume during the loading step, still keep it at 1:10 to 1:100.

Should I use a pump to pass my solution through the column? Maybe I'll just try gravity at first since it is simpler

Troubleshooting Protocol 062912 See Troubleshooting, Problematic Affinity Chromatography for a discussion of elution method.