I do not have direct experience, but I would expect that the amount of your protein in serum will not be that much (compared to usual extracts from transfected/transduced cells/tissues). Everything depends on how sensitive is your Ab and how much protein you expect to be present over there.

I must warn you that when you load serum extract you can rather get a lot of unspecific binding, but that depends a lot on the Ab you use.

I would first run a gel in order to see if you get a clear signal or a signal at all, I would load the maximum amount you can load or different amounts (e.g. 40 µL, 20µL, 10µL for a mini gel with a 10 wells comb, remembering to bring every sample to the highest volume you are loading using lysis buffer for example) to see wherever you are have a signal at lower amounts. The presence of albumin will make everything more "fun" as it is supposed to be highly present and can interfere with your signal.

In case of absence of signal, or too much unspecific one I would go for an IP.

Hope that this can help!

Hi Andrea,

Run multiple lanes of the protein, as used for immunization, on the appropriate % SDS-PAGE. Use various dilutions e.g. 1, 10 and 100 ng protein per lane. Load two or three of such series on one gel. After blotting to membrane (NC or preferably PVDF) mark the lanes, block and then cut the blot in two or three parts. Imuunostaining: serum diluted 1:20 on one part of the blot, 1:100 on another part. If three series were loaded, keep that extra part of the blot in the fridge. Finish the WB and look what results you obtained. If the signal is too strong, dilute further on the extra blot.

Good luck

Hi dear yulia, hope you are fine .

sample preparation

Wash the sub-confluent cells with ice-cold PBS.

Lyse cells with buffer containing 1% SDS and protease inhibitor.

. Homogenize and sonicate the cell lysate. Perform a Bradford or Lowry assay to determine the protein concentration levels.

Add a loading buffer containing SDS and Beta-ME into the whole cell lysate.

Boil the mixture for 5 minutes at 95°C.

Hope this can help you!

Thanks everyone!

Another question that I had is what preparations should be done to blood. Is hemolysis an issue?

Hi Yulia,

Do you meanto ask how to get serum from blood of the mice?

You can treat your blood sample as normal sample which can be lysed e.g. in RIPA (maybe additionaly sonicate your sample for a better protein release). After that you can measure protein concentration and do SDS PAGE, WB etc.

You may want to do immune reactivity just like what we do to confirm vaccine effectivity.

Basically, it's like the traditional western blot but instead of purified antibody, you should use mouse serum.

First, run the protein of your interest or lysate into the gel then transfer to PVDF membrane like WB. Afterwards, incubate the membrane with the mouse serum to allow antibodies it contains to bind on the antigen of your interest. Then detect the binding by treating with secondary antibody with detection enzyme and its substrate.

Don Ashley Olaño Malabana Andreas Eisenreich

Thanks for your answers! That was very helpful.