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Topic Started by Horhay
on 2/1/2005 13:06 PM
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I am currently having a problem with my Negatice Control (Blank), I am seeing absorbance values with my NC corresponding to my lowest standard, the NC is not contaminated, it seems to fluctuate from run to run?
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Replies
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Check the block step. If the plate is not properly blocked, we can hope some background in the negative control. I'm assuming the your blank is treated in the same conditions that your other wells, but without any coat.
Other thing, when we have a "fluctuant" background, it could indicate a problem in the wash step. How are you washing the plates? With PBS with Tween20(0.05%)? Are you using a plate washer or by hands? Did you check if the plates are soaked after you blot in paper towel?
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Posted By Typer
on 5/5/2005 17:09 PM
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Does this require you to block the plate? If so, be sure to follow the recommended times/temperatures/blocking reagent. Some plastics are more "sticky" than others, and work best with specific blocking agents.
If plates are supplied already blocked, be sure that the bottoms or sides of wells are not being touched by plate washer pins or pipet tips during washes, or by pipet tips during a reagent addition. A touch may "deblock" a small area, enough to cause a significant signal increase in a negative control.
Some assays are sensitive to wash buffer retained in the well, especially at the substrate step. It may help to slap the wells a few times against a stack of paper towelling to remove as much wash as possible.
My experience has been that wells allowed to dry out almost always show an increase in absorbence. Another thing to watch out for
These are all pretty obvious things, but sometimes these are the ones we overlook as we try to troubleshoot a malfunctioning assay. Good luck.
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Some other things to consider that can be reason(s) for background signal:
1) Instability of substrate
2) cross reactivity of Ab to irrelevant antigens
3) non-specific binding of Ab to wells (perhaps try another blocking agent...nonfat milk or casein work well, block up to 300ul as well just to make sure each well is completely blocked.
4) non-specific binding of detection agent to wells
5) presence of unexpected anti-Ig in the serum.
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Posted By judy
on 3/28/2006 9:35 AM
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| Tater Salad said: | Some other things to consider that can be reason(s) for background signal:
1) Instability of substrate
2) cross reactivity of Ab to irrelevant antigens
3) non-specific binding of Ab to wells (perhaps try another blocking agent...nonfat milk or casein work well, block up to 300ul as well just to make sure each well is completely blocked.
4) non-specific binding of detection agent to wells
5) presence of unexpected anti-Ig in the serum.
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