I'm relatively new to Western blotting and I've been recently having issues with an extremely dark background on my pvdf membrane. This time I paid close attention to wetting it in methanol (what is a good amount of time to have the membrane sit? I use 0.2 um from Thermo). I prepared my antibodies in new 5% NFDM and probed with my primary for 2 hours at RT and secondary for 1 hr at RT. I was 3x for 5 minutes in between each antibody. My main concern is that we had an issue with my pH meter, so our TBST pH may be slightly off from what it should be, which is 7.4-7.6. I thought this may be throwing off my results, but I can't think to come up with a reason as to why this might be happening. I can't seem to find any other resources describing the problem I'm having and it's quite frustrating. Any input would be greatly appreciated!
Hi,
Sometime, not washing the blot enough can give this kind of problems. I would advice you to wash your blot for at least a total 30 mins (3 changes with 10mins each) after keeping in the primary. washing the blot before addition of secondary antibody may lead to unspecific binding.
II would also suggest you to double check the chemical and the amount you are using to view your blot.
Hope that helps
I may think that was primary Ab is incubated more time or conc used was more, however, have you done blocking before Ab treatment? And make sure that the Ab was prepared in blocking reagent.
In general 45min to 1hr is best for the primary Ab @ RT. (Overnight @4* C)
Use pH litmus paper for alternate (if pH meter was wrong), washing time 3 times and standardize different conc of protein from low to high, antibody dilution also standardize, then you will get very good result in a particular concentration.
Steps involved after transfer gel to PVDF membrane
Blocking>primary Ab>secondary Ab>Development with Chemi-luminescent
Good luck
I'm assuming that protein ladder is in lines 1 and 6. If your protein ladder is compatible with Chemiluminescent detection method it should “shine bright like a diamond [Rihanna]”. Which might suggest that you skipped blocking step as was mentioned earlier and primary antibodies are bound everywhere not only to your protein of interest.
Other possibility
Your ladder is not compatible with chemiluminescent detection and protein isolates are lacking sufficient amount of your desired protein. In result detection system is overexposing your membrane, enhancing even slightest signal he gets from unspecific compounds bound to the background of your WB.
Hi Debadrita,
Thank you for your advice, I'll be sure to try it out! From what I have found, washing is crucial for reducing the background. Do you happen to know mechanistically what occurs during these washes that causes the reduction?
Hi Bhaskar,
I do block for 1 hr at RT with 5% NFDM. I used a 1:1000 dilution for my primary and this duration has worked for me in the past, so I'm not sure as to why it would've given me these faulty results this time around. I think I'm going to pH my TBST then if it is off, I'll make new then redo the rest of my solutions. Thank you for your advice, I greatly appreciate it!
- Badges
- Science method