As suggested by the last speaker this could be non specific binding

• Alternatively, In my exoetience, even with protease inhibitors sometimes it is physiological;  that is a natural degradation product in the cell and thus application of inhibitors designed to limit external degradation will not stop this. Alternatively it can simply be an alternative isoform
• I have also had situations where I have seen multiple bands including putative specific bands with one source of antibody but not snother
• Apart from optimising this is often solved by use of an alternative antibody
• If the lower product is indeed physiological then you can either purchase peptide that was used to elicit the antibody; pre incubate the antibody with such a peptide and then demonstrate a reduction in signal of your expected band but not lower bands - suggesting non specific binding - or both bands suggesting a truncated version of the actual protein; whether a physiological breakdown product or another Isoform
• In addition make another lysate and blot with both your antibody to gene of interest and also housekeeper to ensure you do not see multiple bands: that would suggest external degradation of your lysate. Moreover try not to freeze thaw lysate too many times
• Finally you might want to consider if the above does not work obtaining done lysate of the reference cell line mentioned in your antibody data sheet and ensuring that gives just the one expected band which would imply something incipient and physiological where your own cell line was concerned 
• Consult the literature on your own source material to make sure others have not reported other bands; unlike the ref line

hi, Do you see the 25/30kD band in your other lanes/samples? especially in your negative control? can you upload your Western blot picture here? it is not uncommon to get non-specific band/s in Western blot. You may want to increase the secondary antibody dilution and or reduce the time of antibody exposure.

As suggested by the last speaker this could be non specific binding

• Alternatively, In my exoetience, even with protease inhibitors sometimes it is physiological;  that is a natural degradation product in the cell and thus application of inhibitors designed to limit external degradation will not stop this. Alternatively it can simply be an alternative isoform
• I have also had situations where I have seen multiple bands including putative specific bands with one source of antibody but not snother
• Apart from optimising this is often solved by use of an alternative antibody
• If the lower product is indeed physiological then you can either purchase peptide that was used to elicit the antibody; pre incubate the antibody with such a peptide and then demonstrate a reduction in signal of your expected band but not lower bands - suggesting non specific binding - or both bands suggesting a truncated version of the actual protein; whether a physiological breakdown product or another Isoform
• In addition make another lysate and blot with both your antibody to gene of interest and also housekeeper to ensure you do not see multiple bands: that would suggest external degradation of your lysate. Moreover try not to freeze thaw lysate too many times
• Finally you might want to consider if the above does not work obtaining done lysate of the reference cell line mentioned in your antibody data sheet and ensuring that gives just the one expected band which would imply something incipient and physiological where your own cell line was concerned 
• Consult the literature on your own source material to make sure others have not reported other bands; unlike the ref line

Thank you very much for the answers

I will try to add a picture later. 

Here I add one of the membranes where I get the bands which I am talking about. The lower one

I know that I have low levels of proteins, but the thing that worried me most is the lower band which appears in the last 4-5 lanes.

I have read your comments and I am going to prepare new aliqutos because I have freeze/thaw those samples maybe 5 /6 times. 

Thank you! 

• Yes 5-6 times could lead to some degradation
• However your signal in general is low with some evidence of bsckground
• thus try loading as much as 30-50ug of your lysate with ECL or better still ECL prime but in this case use maybe 5ug of lysate
• ECL prime can be 10 X as sensitive as standard ECL:
• http://www.gelifesciences.com/webapp/wcs/stores/servlet/ProductDisplay?categoryId=11148&catalogId=10101&productId=16746&storeId=12751&langId=-1
• Thus with standard ECL:

• For ECL PRIME

• You might need to play with the above conditions but this gives you a starting point 

Thank you very much Laurence Suart Hall

Tomorrow I will try a new WB with new aliquots. I hope that there is a problem of degradation... we will see...

I am loading a 40 ug of lysate. We use in the lab the "Clarity Western ECL Substrate from Biorad" and we incubate 5 minutes every membrane. 

I am doing O/N 4ºC first antitody and one hour second antibody Room Temperature. In both solutions I use Tween 20 0.1%.

After every antibody I am doing 4 washes of TBST 15 minutes per wash 

I do not know if to change the conditions for my blocking.. I am blocking 1 and a half hour room temperature... 

Thank you very much, I apreciate any more comments.

I will comment if I get better results using new aliquots and changing the second antibody, because it is recommended between 1:1000 and 1:3000 and I have been using 1:2000. I will use 1:3000 tomorrowm Maybe I will try more blocking...

Thank u once again

• There is some evidence that blocking over night at 4C can improve background
• Furthermore addition of 1% BSA to your 3-5% milk powder or use of 2% BSA instead of 2-5% milk powder can reduce background
• However it can sometimes reduce specific signal as well
• Thus I would say block over night at 4C using 5% milk in TBST + 0.1 to 0.2% tween 20. That might help although extend your blotting protocol so maybe just elevate milk to 5% and also make sure you have tween 20 in your blocking regimen as well

On another subject because your bands specific and non specific alike are weak do you make sure that during transfer all of your ladder < 100KD is transferred to the membrane and do you verify lysate protein transfer by transient ponceau S staining ?

• http://www.sigmaaldrich.com/catalog/product/sial/p7170?lang=en®ion=GB&gclid=Cj0KEQjw6uO-BRDbzujwtuzAzfkBEiQAAnhJ0CqpBcVLgeW-eyijMn7Dp9hSDJd6LC_fFFCoa0Gs4CsaAg0l8P8HAQ
• https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Product_Information_Sheet/p7170pis.pdf