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analyte recovery/ligand fishing
- OldForum
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10 years 10 months ago #1
by OldForum
analyte recovery/ligand fishing was created by OldForum
hello,
i am having some difficulties regarding an experiment on a Biacore 3000. I want to recover (in a vial) a protein (73000 Da) that is overexpressed in eukariot cells using antibodies (Ab) immbolized to a CM5 chip.I read some articles, i know that the recovered analyte is in small volumes 2uL, about surface prep unit that it can enhance the recovery due to the bigger area available for immobilization. The lyse of the cells was made in Hepes-CHAPS 2% plus protease inhibitors should i try to keep the analyte buffer as simple as possible?
the difficulties appear in
1) how much Ab should I immbolize (is 10000RU a good start, a conc of 50ug would do)?
2) the analyte will be a cell lysate which will contain the interest protein (i am thinking to centrifuge as a sort of preclear). i did a BCA to see the concentration of the whole proteins in the lysate is 1.5mg/ml. Is 75ug/uL a good concentration to start.
3) should i keep a low analyte concentration and inject over a longer period of time (i'm thinking that way i can reduce the non specific binding if the sample is more diluted)
4) for wash sensor chip solution 150mM NaCl, 50mM NaOH or CHAPS 1% with 100mM NaCl.
5) for elution 10 mM glycine pH 3, HCL, HEPES-CHAPS 2% or SDS 0.5%
thanks!!!!
by the way is the buffer HBS or sensor chip bought from GE (CM5) will expire can you still use it?
Cristi
i am having some difficulties regarding an experiment on a Biacore 3000. I want to recover (in a vial) a protein (73000 Da) that is overexpressed in eukariot cells using antibodies (Ab) immbolized to a CM5 chip.I read some articles, i know that the recovered analyte is in small volumes 2uL, about surface prep unit that it can enhance the recovery due to the bigger area available for immobilization. The lyse of the cells was made in Hepes-CHAPS 2% plus protease inhibitors should i try to keep the analyte buffer as simple as possible?
the difficulties appear in
1) how much Ab should I immbolize (is 10000RU a good start, a conc of 50ug would do)?
2) the analyte will be a cell lysate which will contain the interest protein (i am thinking to centrifuge as a sort of preclear). i did a BCA to see the concentration of the whole proteins in the lysate is 1.5mg/ml. Is 75ug/uL a good concentration to start.
3) should i keep a low analyte concentration and inject over a longer period of time (i'm thinking that way i can reduce the non specific binding if the sample is more diluted)
4) for wash sensor chip solution 150mM NaCl, 50mM NaOH or CHAPS 1% with 100mM NaCl.
5) for elution 10 mM glycine pH 3, HCL, HEPES-CHAPS 2% or SDS 0.5%
thanks!!!!
by the way is the buffer HBS or sensor chip bought from GE (CM5) will expire can you still use it?
Cristi
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- OldForum
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10 years 10 months ago #2
by OldForum
Replied by OldForum on topic analyte recovery/ligand fishing
Hi Cristi,
In my opinion, the experiation date is a more legal thing than that some chemical wil not work any more. Just use it!
1) High Ab concentration on the chip means more protein to recover.
2) Always inject cleared / filtered (0.45 uM) solutions. Proteins can easily pass this kind of filter. Try different concentrations, see 3)
3) The aim is to recover as much as possible protein. So inject a concentration that will saturate the Ab in a reasonable time
4) The washing should wash away non-specific bound proteins. The solutions depends on the strentgh of the iteraction between the Ab and the protein
5) Also, use the solution which elute your protein but keeps it intact and also retains the activity of the Ab. Can be a diffilcult one.
There are no 'one size fuit all' solutuons in this case.
Why do you not make an affinity column with the antibody? In that way you can isolate your protein in much higher quantities and it wil take less time.
You still have to discover the proper purification conditions.
regards
Arnoud
In my opinion, the experiation date is a more legal thing than that some chemical wil not work any more. Just use it!
1) High Ab concentration on the chip means more protein to recover.
2) Always inject cleared / filtered (0.45 uM) solutions. Proteins can easily pass this kind of filter. Try different concentrations, see 3)
3) The aim is to recover as much as possible protein. So inject a concentration that will saturate the Ab in a reasonable time
4) The washing should wash away non-specific bound proteins. The solutions depends on the strentgh of the iteraction between the Ab and the protein
5) Also, use the solution which elute your protein but keeps it intact and also retains the activity of the Ab. Can be a diffilcult one.
There are no 'one size fuit all' solutuons in this case.
Why do you not make an affinity column with the antibody? In that way you can isolate your protein in much higher quantities and it wil take less time.
You still have to discover the proper purification conditions.
regards
Arnoud
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