I conducted an affinity purification experiment. I tested the wash for any unbound Ab and I found a signal in the 0,5 M NaCl wash, and I want to incubate it again with the matrix to catch as much as possible of the unbound antibody. Is this possible?.
Based on your statement, I assume you are trying to capture an Ab using a protein G/A column? If this is true then yes you can dilute your unbound wash down to 0.1M and rerun. However, you should not be seeing your antibody in the 0.5M wash step, unless the column is not binding very well or there is another problem. Are you sure the unbound portion is your antibody? Are you sure you didn't overload the column?
Yes, it's possible, if you dilute 0.5 M NaCl eluted fraction about 10-fold you will decrease the concentration of NaCl to 50 mM and you can load this preparation again onto your column overnight at low flow rate at 4°C under conditions of recirculation. In that case you will be able to catch more of your antibody because of the greater "residence time" for the interaction of your antigen (or Protein G/A) and antibody. This is very important when you have to deal with some low-affinity antibody (in the case of using antigen column) which usually does not bind to the antigen at high concentration of salt (like NaCl).
Dear Daniel, Thanks for your help, yes you are right I used the reagents that came with the protein G column but I used them to purify the specific Abs to a certain synthetic peptide, I confirmed the existence of my Abs in the it was my Abs with ELISA it gives a weak signal, Maybe you are right in the point of overloading, as I used (2ml) of previously purified Abs and incubated them with the agarose matrix (0,25 ml) that was previously coupled to 10 mg of the same synthetic peptide I used it in the ELISA plate coating.
Instead of diluting, you can make a desalting step on a desalting column. With a desalting step you prevent more bleading on your affinity column.
Thanks Nadine for your help., I want to ask about the optimum amount or Conc. of the agarose, coupled peptides and serum for the affinity purification experiment to avoid the overload problem.
- Badges
- Science method
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Methods
- Protein Purification Techniques