serum purification? - (Mar/09/2007 )
Hi!!
I have generated a serum against two peptides. But I don't have the purified antibody. When I make a Western-blot or immunohistochemistry experiments, I have some non specific bands or background.
Do you have protocols in one hand, to purify the antibody from the serum and in the order hand, to exhaust the serum against a cell lysate (to avoid non specific signals).
Thinks for all.
Bye
Aurore
-Auror37-
QUOTE (Auror37 @ Mar 9 2007, 09:00 AM)
Hi!!
I have generated a serum against two peptides. But I don't have the purified antibody. When I make a Western-blot or immunohistochemistry experiments, I have some non specific bands or background.
Do you have protocols in one hand, to purify the antibody from the serum and in the order hand, to exhaust the serum against a cell lysate (to avoid non specific signals).
Thinks for all.
Bye
Aurore
I have generated a serum against two peptides. But I don't have the purified antibody. When I make a Western-blot or immunohistochemistry experiments, I have some non specific bands or background.
Do you have protocols in one hand, to purify the antibody from the serum and in the order hand, to exhaust the serum against a cell lysate (to avoid non specific signals).
Thinks for all.
Bye
Aurore
You have only non specific bands or major band of your peptides occur among them? If you have impurities in your peptides may be you obtain strong immune response to impurities but not for your pep?
To purify the most rapid but not cheap is protein G sepharose, before loading on column filter your serum through glass wool to get rid of lipids
-circlepoint-
QUOTE (circlepoint @ Mar 9 2007, 06:18 PM)
QUOTE (Auror37 @ Mar 9 2007, 09:00 AM)
Hi!!
I have generated a serum against two peptides. But I don't have the purified antibody. When I make a Western-blot or immunohistochemistry experiments, I have some non specific bands or background.
Do you have protocols in one hand, to purify the antibody from the serum and in the order hand, to exhaust the serum against a cell lysate (to avoid non specific signals).
Thinks for all.
Bye
Aurore
I have generated a serum against two peptides. But I don't have the purified antibody. When I make a Western-blot or immunohistochemistry experiments, I have some non specific bands or background.
Do you have protocols in one hand, to purify the antibody from the serum and in the order hand, to exhaust the serum against a cell lysate (to avoid non specific signals).
Thinks for all.
Bye
Aurore
You have only non specific bands or major band of your peptides occur among them? If you have impurities in your peptides may be you obtain strong immune response to impurities but not for your pep?
To purify the most rapid but not cheap is protein G sepharose, before loading on column filter your serum through glass wool to get rid of lipids
I have non specific bands but I can see the major band corresponding to the protein against to! I not sure that I have impurities in my peptides because purity was verify before rabbits' immunization. I suppose that what I see correspond to rabbits' bands. I will try to use BSA instead of milk for blocking the membrane.
Ok for protein G sepharose, I will try too!!!
-Auror37-
QUOTE (Auror37 @ Mar 10 2007, 11:56 PM)
QUOTE (circlepoint @ Mar 9 2007, 06:18 PM)
QUOTE (Auror37 @ Mar 9 2007, 09:00 AM)
Hi!!
I have generated a serum against two peptides. But I don't have the purified antibody. When I make a Western-blot or immunohistochemistry experiments, I have some non specific bands or background.
Do you have protocols in one hand, to purify the antibody from the serum and in the order hand, to exhaust the serum against a cell lysate (to avoid non specific signals).
Thinks for all.
Bye
Aurore
I have generated a serum against two peptides. But I don't have the purified antibody. When I make a Western-blot or immunohistochemistry experiments, I have some non specific bands or background.
Do you have protocols in one hand, to purify the antibody from the serum and in the order hand, to exhaust the serum against a cell lysate (to avoid non specific signals).
Thinks for all.
Bye
Aurore
You have only non specific bands or major band of your peptides occur among them? If you have impurities in your peptides may be you obtain strong immune response to impurities but not for your pep?
To purify the most rapid but not cheap is protein G sepharose, before loading on column filter your serum through glass wool to get rid of lipids
I have non specific bands but I can see the major band corresponding to the protein against to! I not sure that I have impurities in my peptides because purity was verify before rabbits' immunization. I suppose that what I see correspond to rabbits' bands. I will try to use BSA instead of milk for blocking the membrane.
Ok for protein G sepharose, I will try too!!!
Do you check you peptides on tricine PAGE?
-circlepoint-
It's a society that design peptides against the interest protein and realize the antiserum. I don't know exactelly their protocols...
We just don't obtain the purified antibody. A friend of mine advises me to exhaust the serum against cell lysate to eliminate background observed. Do you know this method?
-Auror37-
QUOTE (Auror37 @ Mar 12 2007, 12:30 AM)
It's a society that design peptides against the interest protein and realize the antiserum. I don't know exactelly their protocols...
We just don't obtain the purified antibody. A friend of mine advises me to exhaust the serum against cell lysate to eliminate background observed. Do you know this method?
We just don't obtain the purified antibody. A friend of mine advises me to exhaust the serum against cell lysate to eliminate background observed. Do you know this method?
Do you have enough peptides for making an affinity column to purify your antiserum?
-genehunter-1-
QUOTE (genehunter-1 @ Mar 12 2007, 04:27 PM)
QUOTE (Auror37 @ Mar 12 2007, 12:30 AM)
It's a society that design peptides against the interest protein and realize the antiserum. I don't know exactelly their protocols...
We just don't obtain the purified antibody. A friend of mine advises me to exhaust the serum against cell lysate to eliminate background observed. Do you know this method?
We just don't obtain the purified antibody. A friend of mine advises me to exhaust the serum against cell lysate to eliminate background observed. Do you know this method?
Do you have enough peptides for making an affinity column to purify your antiserum?
[color="#9932CC"][/color]
I'm not sure having enough peptide... I've got 15mg for one and 19mg for the other... Is it ok for affinity column?
-Auror37-