densitometry on western blots

Jose de las Heras via methods%40net.bio.net (by josenet At tiscali.co.uk)
Thu Nov 23 16:05:09 EST 2006


"DK" <dk At no.email.thankstospam.net> wrote in message 
news:gql9h.205$Ll4.132 At newsfe04.lga...
> In article <E3l9h.5384$k6.3071 At bignews8.bellsouth.net>, 
> aawara At FEMA-trailer.org wrote:
>>In <wGk9h.197$Ll4.38 At newsfe04.lga>,
>> DK <dk At no.email.thankstospam.net> wrote:
>>
>>> In article <1164299514.092168.323980 At f16g2000cwb.googlegroups.com>,
>> martinhoehne At gmx.net wrote:
>>>>Hello,
>>>>
>>>>I have some basic questions on densitometry and would be glad if anyone
>>>>can point me to a good source to learn more or even answers some of the
>>>>questions.
>>>>
>>>>1) As I understand it the bands have to be non-saturated. Does this
>>>>really mean that as soon as I have pixel values of 255 (on a 8-bit
>>>>image) I can forget about doing a densitometric analysis?
>>>
>>> No, it does not.
>>
>>That's not right.  For an 8-bit image, once a pixel value is at 255,
>>that pixel value cannot get any higher - by definition it is already
>>fully saturated.  Practically speaking, from an 8-bit image you get
>>two logs of linearity.
>
> Practically speaking, two logs is well within a range biologists
> operate on Western blots. And, practically speaking, any number of
> shades of gray is completely meaningless without a calibration curve.

Having a calibration curve is a very good thing, as long as you're not using 
saturated bands, as the other poster said.

Once you reach saturation... you can't say anything about that sample, only 
that it is high. If you want to do any sort of quantitative measurement, 
even rough, you have to stay within the dynamic range of the capture system, 
i.e: no saturation.

 But I agree that an 8-bit system is enough to give you pretty good results.

Jose 




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