I am using IgY fractions for detecting bacterial proteins. The antibodies are verified to detect the purified protein of interest. When i use the antibodies in combination with cell lysate, significant noise is observed.
I have tried various dilutions for blotting but it does not remove the noise specifically.
I typically use 5% non dairy milk for blocking in 0.1% Tween-20. The blotting is done at 25 degrees/1 hour or cold room/overnight. The washing are done at RT with TBSBT(0.1% TWEEN-20).
I am thinking to try TBST with 0.5-1% Tween-20 for washing and primary antibody incubation at 37 to decrease the noise. I am wondering if anyone has tries such extreme conditions for blotting?
Hi, I used to do 0.1% TWEEN 20. 1% in my opinion is extremely harsh, but you could try a ray of percentage and check what might work better to decrease the noise. Sometimes, the secondary antibody is too concentrated and that increase the noise too much.
Hi!! What do you mean when you say "noise"? More than one band? Black spots on the plot? Because maybe, if you explain your problem with more detail, people can give you better solutions!
Hi Isabel, sorry for the limited clarity on "noise".
Yes, noise means more than one band i.e. non specific binding of the primary antibody.
I suppose the IgY fraction itself may be having some non-specific binders apart from my antibody of interest as i am not using an affinity purified antibody. So the question here may be measures to reduce this binding.
Thanks Rana, I agree, as i too use 0.1% Tween-20 and 1% seems too high.
I have started of with 0.5 % currently. The secondary antibody in dilutions of 1:10,000 also gives the noise but i have confirmed that the noise does not come from the secondary as blots are totally blank when probed with secondary only.
I think its not uncommon to see several bands of your unpurified recombinant protein in the cell lysate, due to aggregation or protein degradation issues. Usually, this products represent a minor population in comparison to your protein of interest. I would suggest loading less material of lysate onto the Western Blot to outdilute this signals (e.g load 5, 2.5, 1 and 0.5 µg protein per lane of your cell lysate). In my experience, increasing washing stringency does not help very much once the antibody has bound.
Good luck !
I faced the same problem, non-specific binding...You can try with reducing primary and secondary Ab incubation time, block overnight and longer t-tbs washes.
Good luck!!
A quick update. I used 37 degrees /30 minutes and 0.5% Tween 20 in TBST.
The harsh conditions do get rid of the background significantly. This clearly comes at the cost of decrease in the overall antibody binding to the protein of interest as well as non specific binders.
Hi Michael, I do agree partly that once bound, the harsh washing might not help much..so we tried the binding at elevated temperatures in sure seems to help. Secondly we are loading around 10-20 microgm of protein in each lane as the protein to interest are very low in abundance.
- Badges
- Science method