Hi, If you have a sample from  K.O. animal or cell line, that can serve as a negative control. Another thing, try first to incubate your blot directly with secondary antibody (skip primary antibody) and then visualize. In this way you can check if the signal is because of secondary antibody (after this visualization, you can reprobe your blot without any stripping). If the signal is obtained only after incubation with primary AB, then your AB is definitely cross reacting. Check for dimers, trimers of your protein and see if the molecular weight of 150 kDa is in multiple of mol. wt of your protein. At last, know the peptide sequence of the antigen against which your antibody was raised and run BLAST. Good luck!

What is the source of your primary antibody  - monoclonal or rabbit polyclonal ? and the secondary antibody?  What protein are you looking at?

Hi, If you have a sample from  K.O. animal or cell line, that can serve as a negative control. Another thing, try first to incubate your blot directly with secondary antibody (skip primary antibody) and then visualize. In this way you can check if the signal is because of secondary antibody (after this visualization, you can reprobe your blot without any stripping). If the signal is obtained only after incubation with primary AB, then your AB is definitely cross reacting. Check for dimers, trimers of your protein and see if the molecular weight of 150 kDa is in multiple of mol. wt of your protein. At last, know the peptide sequence of the antigen against which your antibody was raised and run BLAST. Good luck!

Hi my suggestion is try running the gel with DTT. It might be sometimes immunoglobulin ( Just a suggestion, a common problem when we work with serum). with DTT it will be cleaved and you may not see that band only if its IgG. Good luck.

Hi Noel,

150 Kda surely looks like IgG. In which species your primary and secondary antibodies are raised?

Ram

Detection of endogenous avidin-binding proteins in rat liver cells by transblot and electron microscopy. Haneji T1, Toyama Y, Nagano T. Author information   Abstract

I assumed that the WB was run under the usual denaturing conditions, in which case IgG should have migrated as light and heavy chains, not as a 150k protein. if not, then it is a possibility as inferred by the query about the source (animal) of primary and secondary Ab.

Incubation with your secondary detection system alone may be informative.

If you use at any stage a biotin antibody + avidin peroxidase, the following may be of interest - if not ignore .

Peroxidase-conjugated avidin was used to detect the endogenous avidin-binding proteins in rat tissues. By a transblot method, avidin-peroxidase interacted with proteins of mitochondrial fractions of rat liver with estimated molecular weights of 120,000 and 74,000. The proteins were identified as pyruvate carboxylase (120 kDa, pI 6.4) and methylcrotonyl-CoA carboxylase (74 kDa, pI 7.2) by two-dimensional gel electrophoresis and transblot method. The estimated molecular weight of an additional band (220,000) detected in the cytosolic fraction of rat liver was consistent with acetyl-CoA carboxylase

Hi Ian, Ramakrishnan, Shambhuprasad and Tushar.

I thank you very much for your reply.

Concerning primary anitbodies, I tried mouse monoclonal and rabbit polyclonal antibodies associated with goat anti rabbit, rabbit anti mouse secondary antibodies ... It gives the same result.

I'm looking at a membrane receptor (RTK).

I have no access to KO animal, but I should find cells that do not express this protein

Incubation of the blot directly with secondary antibody was done, no signal

"Check for dimers, trimers of your protein and see if the molecular weight of 150 kDa is in multiple of mol. wt of your protein. At last, know the peptide sequence of the antigen against which your antibody was raised and run BLAST" good clues ... but I don't have any information about the antigen against which my antibody was raised.

WB was run under the usual denaturing conditions (SDS) and DTT is added to samples before loading onto the gel.

Finally, I do not use biotin antibody + avidin peroxidase.

Thanks

Benoit

Try simply adjusting the WB protocol:

1. Try different dilutions of  primary Ab to obtain optimal signal/noise 

2. increase stringency of washes and time of washes: i.e. increase detergent concentration

Theoretically your Ab should have  higher affinity for the target protein therefore non-specific binding should be reduced with more stringency

I suggest you to try blocking overnight with 5 % BSA ( If you are using milk change to BSA). How long do you incubate with the primary and secondary?

One more thing DTT as you know denatures the protein and it changes the migration pattern. It happens with the family of proteins I work usually. though we do not know the reason exactly. Just do a comparison between non-denaturing and denaturing conditions. I have recent  experience my protein in non denaturing condition detected at 62 and in non denaturing at 95 kDa in denatuting!! (we always do with non- denaturing conditions). But I feel its better check for this possibility. Good luck

Hi Shambhuorasad,

Yes, I'm working in 5% BSA, Saturation = 1h. Primary antibody overnight. Secondary antibody = 1h.

It's a good idea to compare with and without DTT.

Thanks

Try this: load a gel full of liver samples at about 100ug/lane, and blot as usual.  Make up your antibody in 5% filtered BSA in TBST with azide, then leave your antibody on the membrane over the weekend at 4C.  Remove the antibody and try it on a normal western and see if you are getting the cross reactive band on your blot.  This process really cleans antibodies up, especially non specific binding, but also cross reactive binding as well.  Is your antibody a monoclonal or polyclonal?

good luck!

Andy

Hi Noel,

May I ask, which RTK you are looking at and it's expected molecular weight? Many RTKs have some homology. Also, I know you have said you don't have any information about the epitopes of the primary abs you've tried, is it at all possible to obtain this information? It seems unusual for this sort of info to not be available. Were any of the primary antibodies you tried commercial ones? Are you also seeing the protein of interest at the expected molecular weight when you blot? If so, how strong is the signal compared with that of the 150KDa protein?

The idea of trying to remove non-specific Ab binding is in theory a good one. However for this you will need a source of tissue that does not express the RTKs you are trying to detect. This may be a more challenging proposition (cell fractionation? membrane vs non membrane extracts?). I know nothing about RTKs (like most things in life) other than they cover a large family, can form dimers and possess Ig-like regions (thank goodness for Wikipedia). Is there recombinant  RTK available to 1) check Ab reactivity 2) preabsorb your polyclonal - any subsequent signal would be non-specific which you could then at least eliminate/identify.

Also liver is likely to be rich in lymphoid cells which may be a problem with secondary Ab detection systems.

Also try including a non-specific blocking serum  eg Horse serum, Donkey serum, chicken etc etc  to see if it improves the target:non-target discrimination.

must stop spouting nonsense....