polyA RNA staining for dot blot - (Nov/17/2014 )

Hi,

I recently did a dot blot to quantify the m6A polyA RNA levels. For this, after immobilizing the membrane, I did a regular western blotting (block membrane with milk) and then I incubated it with m6A antibody. After finishing the western blot, I'd like to normalize the RNA levels and found that most of the scientist do it with methylene blue. However, when I stain the membrane with methylene blue, it is completely blank. I also stripped out the membrane, just in case the milk and blotting antibody were blocking the methylene blue action. Any suggestion?

Thanks!!!

Hi, 

I actually have a question for you, since I am doing the same procedure as you are with the m6A antibody. 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. If you have some advice I am all ears! Please let me know!

thank you!

purplehayes on Sat Apr 11 15:39:44 2015 said:

Hi, 

 

I actually have a question for you, since I am doing the same procedure as you are with the m6A antibody. 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. If you have some advice I am all ears! Please let me know!

 

thank you!

we found that the best way to deal with this problem is to seal off wells that have dried.

mdfenko on Mon Apr 13 11:21:09 2015 said:

 

purplehayes on Sat Apr 11 15:39:44 2015 said:

Hi, 

 

I actually have a question for you, since I am doing the same procedure as you are with the m6A antibody. 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. If you have some advice I am all ears! Please let me know!

 

thank you!

we found that the best way to deal with this problem is to seal off wells that have dried.

 

What do you use to seal them off? I've heard of using 3% gelatin or tape. 

if it is a dot blot rather than a slot blot then you can use stoppers (cork or rubber). for a slot blot you can use one of those stick on seals (like used to seal elisa plates).