My western blot protocol was :
- Sample:normal human epidermal extract, 25 ug/lane
- 7.5%PAGE, semi-dry transfer to nitrocellulose membranes.
- Blocking buffer:5% skim milk in TBST(0.5% TWEEN 20)
- 1st antibody:Human serum 1:20 diluted in blocking buffer,4 degree ,O/N
- 2nd antibody:Rabbit anti-human IgG（1.3 mg/ml )1:40000 diluted in blocking buffer,RT,1h.
- Washing step: TBST(0.5% TWEEN 20),10 min,2 times;5min,1time.
- ECL prime detection.CCD camera expose.4 sec.
loading pattern:
- lane a and lane b: human serum.
- lane c: without 1st antibody(human serum).
I attach the result,as you can see ,the background from serum was dark,and My western blot was aimed at the high M.W. between 250 kDa to 100 kDa.
I expected lane a had bands between 250kDa to 150kDa, lane b was set as normal control, and there was a strong nonspecific band .
I have tried change blocking condition at 4 degree O/N;change blocking buffer (5%BSA);diluted 1st antibody(human serum) further ,while further dilution weaken the specific banding,and those can't help me to get expected result.
Is there any advice about reduce the background caused from human serum?
Thank you for your help!
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
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