Hi Tie,

have you tried to reduce the protein concentration per lane? reduce to 5 to 10 µg.

You could also add 0.01% SDS in your first Ab, this can reduce the unspecific bindings.

The problem with serum is that you have a lot of other Ab, which might have cross reactions

you could try to purify your Ab by afinity purification,

best regards

Bogdan

Dear Tie,

As you are interested in a 100-250 kDa protein you might consider using a different percentage of SDS-page gel. Try a 5% instead. Your protein of interest will have a higher resolution.

That won’t solve the background. Let me suggest something else for that.

Some serum component does a-specifically stick to the membrane. I do prefer PVDF over nitrocellulose, but I’m not sure that will solve this case.

You could include normal Rabbit serum to the first blocking step. Do not include it in the other steps. Overnight blocking, as you mention, is a good idea.

Less lysate, as mentioned above, will not lead to better results. It does not lower the BG but will lower the specific signal.

An overnight incubation of the 1st Ab is not a good idea. Keep it to 1 hour RT. You may prolong the washing steps.

You show a normal serum control in B. You wonder what the 220 kDA and the >250 kDa band is, isn’t it? Why don’t you test two other normal human sera from other individuals?

A higher % Tween will lower the background too.

Good luck!

Duco Kramer

Thank you Bogdan, you can call me DUERNA.

For you 3 advices,

        As for the cross reaction you mentioned,  75% of IgG in serum supplied a lot of opportunity. Should I trying recombinant antigen as subtract ?

      Thank you very much !

      Best regard!

     DUERNA

 Dear Duco Kramer,

I was think about trying different blocking buffer too!

Normal rabbit serum! I will try to use it !

Just as you say ,test other normal human sera from other individuals to set the normal control, and I have done that ,there were none non-specific band.(attach picture).

It's really hard to identify the lane b's non-specific band since I have tried several times to check lane b's serum ,and there were alway's a band nearly 250kDa. The serum's donor was quite healthy!

Thank you for all the suggestion! 

    Best regards!

   DUERNA

Hi Duerna,

using recombinant antigen as a substrate is a good sugestion

best regards

Bogdan

Hi Duerna

So if I understand it correctly this >250 kDa band is only seen in the serum that was used in your figure 1, lane b. This is not an a-specific band. It is an unexpected band! Maybe the person who donated blood has antibodies against an epidermal protein >250 kDa. Interesting, but I wouldn't use it in this assay. One of the other two sera are just better for your test.

Once you know what affinity purified Ab does (patient lane a and both normal sera from figure 2), you might consider to affinity-purify the serum of 'serum b' too.

Good luck w/ the affinity purification.

Duco

Dear Bogdan

     Thank you Bogdan! 

     Recombinant antigen is not a easy work ,although there are many scientisit have already  done that work (I mean the antigen from epidermal ,such as bullous pemphigoid antigen ,desmoglein and so on), while building a recombinant protein producing system will cost a lot of time . May be this will increase the specific band (because enough low abundance protein) ,but I don't know whether it will reduce the background from human serum or not.

    Trying WB in normal epidermal extract condition is still important to identify antigen, and thank you for you suggestion !

   Best regards!

   DUERNA

Dear Duco

       My next plan was Affinity purification,using IP（pathogenicity IgG) or just  separate whole IgG.As your suggestion ,I will consider affinity-purify the lane b's serum.

       As for the rabbit serum blocking test:

       I have no ready-to-use normal rabbit serum ,but I have normal donkey serum ,and my secondary Abs have no cross-reacts with donkey serum by rocket immunoelectrophoresis, so I tried it .

         I applied 1% donkey serum for blocking ,the background were much clear than other blocking( BSA,Skim milk,PVP, some blocking product from company) when expose for 4sec.But the blot still not suitable for longer time expose that bring's nonspecific band from 2nd Abs.

    I guess there are something(such as amino acid,AA )in human serum and contain to the membranes,and 2nd Abs recognized them and bring the background.Using rabbit or duckey ' serum blocking those aperture in membranes ,instead of human serum's component to fulfill the aperture, this reducing the background.

 Thank you for you advice!

  Best regards!

  DUERNA