I’m using a Western blotting antibody that that worked with samples from human plasma. How can I extract protein from blood plasma? Or is it possible to perform WB using the plasma directly?
Thanks in advance.
Dear Aljouhra,
Considering that the protein concentration in plasma/serum is approximately 60–80 mg/mL, I believe you may try it without further protein extraction. However it will depend on the representation of your protein of interest in the mix. Albumins will make roughly half of the total protein content and globulins will be about one-fifth of the total.
Protein Levels in Human Plasma
PROTEINNORMAL RANGE (mg/dL)
Albumin 3,500 - 4,500
Prealbumin (Transthyretin) 10 - 40
α1-Globulins 300 - 600 α1
Acid Glycoprotein (orosomucoid) 55 - 140
α-Fetoprotein approx. 0.001
Retinol Binding Protein 3 - 6
Thyroxine Binding Globulin 1 - 2
Transcortin 3 - 3.5
α2 Globulins (excluding lipoproteins)________ 400 - 900
Ceruloplasmin 15 - 60
Haptoglobin Type 1-1 100 - 220
Haptoglobin Type 2-1 160 - 300
Haptoglobin Type 2-2 120 - 260
α2 Macroglobulin 100 - 280
β-Globulins (excluding lipoproteins) 600 - 1,100
C-Reactive Protein < 1
Hemopexin 50 - 100
Microglobulin approx. 0.2
Transferrin 200 - 320
γ-Globulins 700 - 1,500
Which plasma protein/s do you want to extract and detect using western blotting..?
As there are too many proteins in plasma, perhaps purifying the protein/s of interest would be a good idea if you have sufficient plasma volume for purification..
After purification, the samples will become much cleaner with perhaps albumin out of the way thus helping for the western blotting - perhaps given less background.. If you still see background them maybe doing a serial dilutions and run all samples side by side for western blotting - perhaps this will help reduce the background due to too many proteins in the sample.
Instead of western blotting you can try ELISA following dilution of your plasma sample.
Dear Aljouhra,
Western blotting on plasma samples works only if your target protein is abundant. It may not work for proteins with low expression.
Alternatively you may try salting out methods (ammonium sulphate) to separate proteins on their molecular weight, dialyse and run on gel.
Good luck,
Sabarinath
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