Hi, what does your gel look like? Are IgGs an Albumin gone? Otherwise you'll need several rounds of clean-up. What is the concentration of the fraction you get ?

If you know the sequence of your protein you should be able to calculate the pI.  This should enable you to use an additional double-negative affinity chromatography step to remove other proteins (apart from the IgG and albumin that you have already tried to deplete) that are charged at the pH where your protein is uncharged (iso-electric point) by running the  serum sample through Q & S sepharose or similar ion exchange resins to maybe deplete the contaminants.  This may risk precipitation of your protein, particularly as you may also need to dialyse to reduce salt concentration to a level where proteins would bind the IEX resins, but given your existing problems it may be a necessity.

Also - which column and conditions are you using to remove the IgG and albumin?  

Hi Markus and Robin,

Thanks for the answers.  I used a IgGs and Albumin removing kit from minipore(122642 | ProteoExtract® Albumin/IgG Removal Kit). I followed the instruction(briefly dilute serum 10 fold with a buffer first, then pass through a beads for the removing, then concentrate to primary volume) and did not check whether IgG and Albumin all gone or not. The protein concentration is about 30-50ug/ul after concentrated, always I load 5-10ul in each test. I did 5 times, 2 times I got a specific band, two times high background and one time no band. The pI suggestion is good, however, we don't have the affinity chromatography column. I don't think it's a prolem from high salt in my sample, beacuse I already dilute it and then concentrate through 3kd-filter. I think the high background maybe result from the high concentrated protein, ie, the target protein has a low abundance, I load too much  total proteins. So, what  do you think?

The idea that your protein is too concentrated after loading sounds plausible based on your figure of 30-50ug/ul which equates to 30-50mg/ml.  This sounds extremely concentrated, particularly if your antibody is sensitive!

Try performing a serial dilution of your protein sample after Ig / Albumin depletion (100%, 10%, 1%, 0.1%, 0.01%, etc..)  ; load equal volumes in adjacent lanes and then perform your western blot again.  If your idea that the concentration is the problem is correct, then maybe you will see clear bands at the higher dilution levels, where the lower dilutions will still give low quality bands.

Another possibility is that your blocking is insufficient in the WB - what blocking agent and % concentration are you using for the primary antibody binding & subsequent WB protocol?  Do you have any detergent present in the washes?  What is the salt concentration?

Good luck!

Hi Robin,

Thank you for help me. Dilution might be a good way. I will try 1/2 to 1/10, as my protein abundance is low. I used 5% mik buffered in 0.1% Teween PBS(PBST). First antibody diluted in  5% mik buffered in 0.1% PBST and incubate overnight at 4 centigrade. Secondary antibody also buffered in 5% mik buffered in 0.1% PBST and incubate 2h.

Salt concentration I think it is similar as the dilution buffer used in the IgG and Albumin removal procedure(I diluted my sample 10 fold using this buffer, and concentrate). Usually, It was physiological.

Thanks!

Dear colleagues,

I have a question that may be relevant to this topic. I am doing also Western Blot on mouse serum. I know that ELISA would be maybe a better choice but this was some sort of not my decision to choose Western Blot instead of ELISA:). I have also problem with albumins giving not specific, strong bands between 50 and 70 kDA which makes impossible to detect my protein of interest namely Angiopoietin 2 which should give a signal around 50-57 kDA. As kits for albumins removal may not be efficient enough Abcam recommended me using pre-absorbed secondary antibody (ab7090). In case of this antibody not specific reactions with proteins in mouse tissue should be strongly reduced. Have you got any experience with that kind of secondary antibody? I would be grateful for your answers/ideas:)

Hi Ewa,

I've used pre-adsorbed Abs many years ago in the context of 'mouse on mouse' antibody kits for immunohistochemistry etc. It worked o.k - certainly enough for a lab experiment. I'm certain that anything being produced now is probably more effective than what I was using back in pre-history. One obvious question I have - is it possible to use a primary antibody from a different species, or is your Angiopoeitin-2 by necessity a mouse monoclonal? Secondly, can you maybe batch-incubate your samples with a Cibachron-blue-based affinity resin to deplete albumin by adsorption before taking the sample for the gel?

Good luck!

Dear Robin,

thank you very much for your answer. My primary antibody is rabbit polyclonal (PA5-27297). In our lab we have not worked with Cibachron-blue-based affinity resin at all. I found that ThermoFisher has a kit: Pierce™ Albumin/IgG Removal Kit based on this reagent, however it is indicated it works with human plasma (and also monkey, swine, rabbit). Have you maybe worked with mouse plasma and Cibachron-blue-based affinity resin? If not maybe we will just have to try it out:)

Thank you one more time for your answer,

Greetings,

Ewa

No problem!

Dye affinity resins and in particular Cibachron are so far off most peoples' radars that it is always nice to bring it up as a topic of discussion - removing unwanted and abundant housekeeping components of serum is a constant problem (particularly with proteomics experiments) and when it is possible to use a slightly old-school method that can work just as well as expensive depletion kits it is worth looking into.

There is a body of fascinating but largely forgotten literature from the late 1970s and early 1980s comparing binding of CB and other related dyes with different families of albumins, some of which could be worth re-investigating. All I can suggest if you are able to obtain the reagent is to try it and see if it works for your experiment.

Best of luck, Robin