western blot quantification - band quantification (Mar/29/2006 )

my protein band show difference at 30 secs but visually the difference vanishes at 1 min. Is signal saturation possible so quickly. What should I believe- 30 secs or 1 min?

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You can get bleeding and saturation very quickly if you have a strong signal. The best quantifiable results are taken at the shortest sensible exposure time.

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hi,
if u r signal is so fast i donot think u can rely on those results(because either 30sec signal is too strong and 1min signal is saturated). try to increase primary and secondary antobody dilutions, i m sure u get nice singnal in sensible time that is around 2-5 minutes.

gud luck

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You can easily get saturation that quickly, I have seen it saturated so much that I have ghost bands...In addition if you continue to have problems even after diluting your antibodies...I found Tropix's kit to be very useful as it gives a graded production of light unlike Pierce's products which ramp up light production very quickly, Tropix gives light over time, so that the strongest signal is definitely at the later timepoint. A rare protein in my samples didn't show up sometimes till 30mins...same samples with Pierce's would show up in 1min

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hi viper,
if i am not wrong, gh100 must be looking for single protein in his western blot as his primary antibodies are specific (although often we see strong non specific bands).
as i mentioned in my previous post, not only primary and secondary antibody, even he can try to dilute his substrate (ECL or pierce substrate) futher 1:5. this would further help to reduce the band intensity ultimately avoids gohst bands. once he standardize the amount of protein to be loaded, antibody and substrate dilutions, he does not have to worry in future with his protein of interest.

if he does not optimized these conditions for his protein of interest then he will use high amounts of antibody and results in bad signal (saturated signals). moreover he must get another way to reduce his band intensity (for example using Tropix substrate) which may not be quite ideal situation.

anyway i would like to request you for information about TROPIX system,
"Tropix gives light over time, so that the strongest signal is definitely at the later timepoint. "
i m curious to know the mechanism behind tropix, would it be possible to help me in understanding its action based on different protein intensities?.

thanks

if u r signal is so fast i donot think u can rely on those results(because either 30sec signal is too strong and 1min signal is saturated). try to increase primary and secondary antobody dilutions, i m sure u get nice singnal in sensible time that is around 2-5 minutes.

gud luck

[quote name='gh100' post='46064' date='Mar 29 2006, 11:29 AM']
my protein band show difference at 30 secs but visually the difference vanishes at 1 min. Is signal saturation possible so quickly. What should I believe- 30 secs or 1 min?
[/quote]
[/quote]

You can easily get saturation that quickly, I have seen it saturated so much that I have ghost bands...In addition if you continue to have problems even after diluting your antibodies...I found Tropix's kit to be very useful as it gives a graded production of light unlike Pierce's products which ramp up light production very quickly, Tropix gives light over time, so that the strongest signal is definitely at the later timepoint. A rare protein in my samples didn't show up sometimes till 30mins...same samples with Pierce's would show up in 1min
[/quote]

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