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Do you need to heat the BSA before 1st use? - (Jun/10/2010 )

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A colleague of mine, who uses a relatively pure BSA (it's tested for DNAse, nuclease etc).. still heats it up at 90 degrees for 15mn to be sure to "kill" any enzyme that might be active in there when he first uses it for molecular biology work.
Another colleague of mine told me that heating the BSA in a heat block when you need to thaw it seems to decrease its efficiency and says it's better to let the BSA thaw at room temperature.

So I'm wondering if heat is a good or a bad thing here and whether I should do the pre treatment at 90. Can it do any damage?
Any thoughts?

-Maddie-

Maddie on Jun 10 2010, 05:40 PM said:

A colleague of mine, who uses a relatively pure BSA (it's tested for DNAse, nuclease etc).. still heats it up at 90 degrees for 15mn to be sure to "kill" any enzyme that might be active in there when he first uses it for molecular biology work.
Another colleague of mine told me that heating the BSA in a heat block when you need to thaw it seems to decrease its efficiency and says it's better to let the BSA thaw at room temperature.

So I'm wondering if heat is a good or a bad thing here and whether I should do the pre treatment at 90. Can it do any damage?
Any thoughts?

I am first time listening this. As far as I know heating of BSA will loose its efficiency. All the commercially available BSAs are quite pure. So there is no need to heat it at 90 degrees.
cheers!!
:lol:

-Enzy-

Thanks Enzy :o

-Maddie-

Never heard of it neither, and have been using BSA for like 5 years without heating it, no problem.

-medchemgirl-

I'm thinking that maybe this colleague used to buy a BSA that wasn't pure and tested for DNAse and got a habit that he just kept until now.

-Maddie-

Yeah you should have seen a decrease in activity when BSA is heated to 90 degrees it undergoes a irreversible strucutral change when its heated known as Heart -> Cigar. This was well charchterized by CD and FTIR during the 80's and early 90's when BSA was hot subject as a wheat additive.

-jerseysurfer66-

Heart to Cigar? :lol:

-Maddie-

I guess he refers to BSA structure...

-hobglobin-

Nice site hobglobin, thanks.

It does describe the action of heat.

Serum albumin when heat-treated, goes through two structural stages. The first stage is reversible whilst the second stage is irreversible but does not necessarily result in a complete destruction of the ordered structure (Kuznetsow et al., 1975; Lin and Koenig, 1976; Oakes, 1976). Heating up to 65C can be regarded as the first stage, with subsequent heating above that as the second stage (Wetzel et al., 1980). The onset temperature of conformation change as found by DSC was 58.1C (Poole et al., 1987) and the temperature of denaturation 62C (Ruegg et al., 1977). Results from CD and IR spectroscopy indicated that beta-sheets were formed when albumin was heated above 65C ( Wetzel et al., 1980), 70C (Lin and Koenig, 1976; Clark et al., 1981b). The beta-sheet formed was more pronounced on cooling and was concentration dependent. Wetzel et al., (1980) found a shoulder in the beta-sheet band at a concentration of 50mg/ml at 70C, 1.4mg/ml at 80C but not for 0.5mg/ml at 75C. Because beta-sheet structures were not indicated in the dilute solution this suggests that the beta-sheets are intermolecular.


However, since nobody knows how BSA works during PCR to reduce inhibition from humic acids or hematin, isn't it a possibility that the BSA works better in its denatured (beta sheet) form? Just wondering since the tubes are heated to over 92 degrees during amplification.

-Maddie-

my colleagues also heat it, but I never saw any difference with or without heating.

However it depends for what purpose you want it for

-Curtis-
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