I am currently working on nucleic acid lateral flow assay. I have tried different buffers that includes formamide, dextran sulfate, denhardt's solution, SSC buffer, PBS buffer in different combinations. But still didnt get recognition on test band. Can anyone help me out in getting rapid recognition at test line.
hybridisation depends on a few factors
Concentration....higher concentration is quicker hybridisation so keep the volume low and further decrease the effective volume of solution with addition of dextran sulphate.Higher temperature ( but below melting temperature) will speed up hybridisation.
I would hybridise at high salt then wash at stringent conditions so hybridisation is quick and dirty but the non specific binding should wash off quickly. Until you have a positive answer leave out the formamide and do not do the high stringency wash. Get a positive result and then clean it up in case the probe is washing off due to too high stringency
hybridisation depends on a few factors
Concentration....higher concentration is quicker hybridisation so keep the volume low and further decrease the effective volume of solution with addition of dextran sulphate.Higher temperature ( but below melting temperature) will speed up hybridisation.
I would hybridise at high salt then wash at stringent conditions so hybridisation is quick and dirty but the non specific binding should wash off quickly. Until you have a positive answer leave out the formamide and do not do the high stringency wash. Get a positive result and then clean it up in case the probe is washing off due to too high stringency
Prefer using nylon 66 membrane instead of nitrocellulose, stringent condition is referred by Paul are worth considering.
In addition, I would recommend testing all of your components at higher concentration first. Prove to yourself that you can see 'easy' signal, just to be certain that you are not working near or below your detection threshold. Start by printing a high concentration of capture oligo and flow a high amount of control analyte -- once you can detect the high signals, you'll have more confidence to move your assay toward higher sensitivity. And, indeed, the suggestions above are extremely appropriate.
Separately, during a microarray project I was working on, we were having trouble with low/undetectable signal. One of the topics was that we were working on was the immobilization chemistry for the capture oligo. It turned out we had made such aggressive chemistry that the oligo was actually so densely packed onto the surface that the 'analyte' oligos couldn't bind. Simple steric hindrance. What seemed counterintuitive was the solution: greatly reduce the concentration of the oligo being printed, and the hybridization signal went way up. The lesson may apply here: maybe it's not your hybridization conditions that are stopping you from getting good data.
Also you can check if the capture oligo is prone to secondary structure formation, and the complexity of the target may also be relevant (C0T value). In general I would try to hybridize at Tm-10degC. Using a dilution series of complementary and non-complementary oligo might help to troubleshoot and identify the problem. As mentioned above start working from a positive signal...
Thank you for all the reply. It is of great use. I will troubleshoot using dilution series of oligo.
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