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Dot blot anomaly - (Dec/22/2013 )

I carried out a dot blot for various concentrations of my cell lysate and found weak positive staining for my protein of interest at a concentration of 15-25ng. However there was absolutely no staining at higher lysate concentrations (the positive control stained even at 100ng). Can someone tell me why? Thanks!


was the positive control a lysate or purified protein?


The positive control was also a whole cell lysate




I am new to this forum and I have a question. My question regards the actual suctioning of samples onto the membrane using a dot blot apparatus. Have you had any difficulty getting the samples to properly suction from the wells onto the membrane? Everything will suction just fine other than the samples themselves (fairly highly diluted RNA). I have gotten this to work with inconsistent results correlating with variable rate of suction speeds. I have been hydrating the membrane prior to dot blot apparatus assembly, full suction during assembly, have used filter paper underneath the membrane, have not used filter paper under the membrane, have used buffer (DEPC water) prior to sample application for membrane rehydration, have used buffer for unused wells to increase suction in the sample wells, have elevated the entire apparatus above vacuum level, and the troubleshooting list goes on. I have been getting diffuse signals indicating improper suctioning. If you have some advice I am all ears! Please let me know!


thank you!