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Help_Role of BSA for the protein reconstitution


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#1 jhkimpko

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Posted 30 June 2009 - 08:44 AM

Hi,

I use rhPDGF-BB and incorporate it into the drug carrier that we developed and measure its release kinetics. However, when rhPDGF-BB (that comes in as a powder form in a glass vial) is reconstituted, addition of 0.1% of BSA (w/v %) is recommended by the manufacturer to prevent non-specific binding of rhPDGF-BB to the inside surfaces of the vial. It's my best interest not to have BSA because it seems like BSA interrupts the release of rhPDGF-BB when it co-exists with rhPDGF-BB in the drug carrier.

I'm wondering,
1) what is the role of BSA? Does its presence really prevent non-specific binding? If this is the case, what is the mechanism?

2) if I'm not adding BSA when rhPDGF-BB is reconstituted, roughly how much loss (%) of rhPDGF-BB is expected when rhPDGF-BB is transferred to another "low binding" vial from an intial glass vial?

3) whether there is any "good" way to take out rhPDGF-BB from an inital glass vial without using BSA?

This is a really significant question that needs to be addressed, and I really wish someone can answer these. Thanks a million!!!

Jay

#2 bob1

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Posted 30 June 2009 - 04:22 PM

The BSA just acts as a blocker, much like in a western blot for the. I don't know how much compound you are likely to lose by not dissolving in BSA. My only suggestion for the getting it out of the vial would be to transfer it as a powder, but then you would lose quite a bit too.

#3 klinmed

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Posted 30 June 2009 - 09:43 PM

Hi,

I use rhPDGF-BB and incorporate it into the drug carrier that we developed and measure its release kinetics. However, when rhPDGF-BB (that comes in as a powder form in a glass vial) is reconstituted, addition of 0.1% of BSA (w/v %) is recommended by the manufacturer to prevent non-specific binding of rhPDGF-BB to the inside surfaces of the vial. It's my best interest not to have BSA because it seems like BSA interrupts the release of rhPDGF-BB when it co-exists with rhPDGF-BB in the drug carrier.

I'm wondering,
1) what is the role of BSA? Does its presence really prevent non-specific binding? If this is the case, what is the mechanism?

2) if I'm not adding BSA when rhPDGF-BB is reconstituted, roughly how much loss (%) of rhPDGF-BB is expected when rhPDGF-BB is transferred to another "low binding" vial from an intial glass vial?

3) whether there is any "good" way to take out rhPDGF-BB from an inital glass vial without using BSA?

This is a really significant question that needs to be addressed, and I really wish someone can answer these. Thanks a million!!!

Jay

Glass and plastic surfaces (polystyrene especially) bind protein in a non-specific manner. The interaction is both hydrophobic and ionic.
High concentrations of carrier protein block these binding sites.

During the early days of cytokine research the extreme losses of activity during purification caused major headaches. This was found to be due to non-specific binding to chromatography columns/tubing etc. One solution was found to be very effective-the addition of 0.02% Tween 20 to all buffers. In addition, whenever possible polypropylene tubes were used.

Some groups used 0.02 % PEG 8000 in a similar manner (not as effective as Tween).

Remember also to keep the ionic strength above 100 mM to minimize ionic interactions.

Hope this helps

#4 jhkimpko

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Posted 01 July 2009 - 06:38 AM

Hi,

I use rhPDGF-BB and incorporate it into the drug carrier that we developed and measure its release kinetics. However, when rhPDGF-BB (that comes in as a powder form in a glass vial) is reconstituted, addition of 0.1% of BSA (w/v %) is recommended by the manufacturer to prevent non-specific binding of rhPDGF-BB to the inside surfaces of the vial. It's my best interest not to have BSA because it seems like BSA interrupts the release of rhPDGF-BB when it co-exists with rhPDGF-BB in the drug carrier.

I'm wondering,
1) what is the role of BSA? Does its presence really prevent non-specific binding? If this is the case, what is the mechanism?

2) if I'm not adding BSA when rhPDGF-BB is reconstituted, roughly how much loss (%) of rhPDGF-BB is expected when rhPDGF-BB is transferred to another "low binding" vial from an intial glass vial?

3) whether there is any "good" way to take out rhPDGF-BB from an inital glass vial without using BSA?

This is a really significant question that needs to be addressed, and I really wish someone can answer these. Thanks a million!!!

Jay

Glass and plastic surfaces (polystyrene especially) bind protein in a non-specific manner. The interaction is both hydrophobic and ionic.
High concentrations of carrier protein block these binding sites.

During the early days of cytokine research the extreme losses of activity during purification caused major headaches. This was found to be due to non-specific binding to chromatography columns/tubing etc. One solution was found to be very effective-the addition of 0.02% Tween 20 to all buffers. In addition, whenever possible polypropylene tubes were used.

Some groups used 0.02 % PEG 8000 in a similar manner (not as effective as Tween).

Remember also to keep the ionic strength above 100 mM to minimize ionic interactions.

Hope this helps


Thank you all for the comments. It was really a valuable information. Certainly, I will try. Again, thanks.

#5 jhkimpko

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Posted 01 July 2009 - 07:04 AM

My follow-up question is then, according to the manufacturer, it is recommended that rhPDGF-BB is reconstituted in 4mM HCl. Would there be any side-effects then by using more concentrated HCl such as 100mM (as you suggested) (ie. on stability of the protein)?

#6 klinmed

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Posted 01 July 2009 - 07:29 AM

My follow-up question is then, according to the manufacturer, it is recommended that rhPDGF-BB is reconstituted in 4mM HCl. Would there be any side-effects then by using more concentrated HCl such as 100mM (as you suggested) (ie. on stability of the protein)?

A simple rule is: Unless absolutely necessary ALWAYS follow the manufacture´s instructions.
There may be sufficient salt from the freeze-drying for example.

Would suggest you dissolve in 4 mM HCL + Tween 20 (if you must have carrier free) at >50 ug/ml. You can make further dilutions in buffered saline (>100 mM NaCL + Tween 20)

Hope this helps.




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