Hi Namita,

By all means, the protein of your interest has a membranous nature and belongs to peripheral blood mononuclear cells. If you are interested in purifying  proteins of lymphocytes, macrophages/monocytes), you must purify your PBMC using the following density gradient procedure. Lysing Red blood Cells: Many commercially available reagents are available that lyse RBCs. Most are simply ammonium chloride solutions. RBC lysis buffer can easily and inexpensively be made: 10x RBC Lysis Buffer 90 g. NH4Cl (0.155M) 10 g. KHCO3 (0.01M) 370 mg. EDTA (0.1mM) Dissolve in one liter of ddH2O Filter sterilize through .22 micron filter. Dilute 1:10 in ddH2O before use. RBC may be lysed before or after staining. Mix 200 µl of whole blood with 2 ml of lysis buffer, incubate at room temperature for 5 minutes, spin down at 300 x g in order to remove lysis buffer. Repeat if necessary. The cells are ready for analysis or staining. Note, Namita, - leukocytes should not be left in lysis buffer for extended periods of time, as cell morphology and viability will be negatively affected. Now on the major procedure - PBMC protein isolation: Human PBMC are isolated using a density gradient technique. The two most commonly used density gradient solutions are Ficoll-Paque PLUS from GE Healthcare Life Sciences and Histopaque- 1077 from Sigma Aldrich. The separation can be done in either 15 or 50 ml conical tubes. Please, obey the following steps. First, aliquot 1 ml of Ficoll-Paque PLUS or Histopaque-1077 for every ml of peripheral blood to be processed. Let Ficoll-Paque PLUS or Histopaque-1077 warm to room temperature as density is temperature-dependent. Second, mix 1 ml of blood with 1 ml of a balanced salt solution, normally PBS. Third, overlay the mixed blood solution on top of the Ficoll-Paque PLUS or Histopaque-1077. Ratio should be 2 ml of the mixed blood + PBS solution for every 1 ml of Ficoll-Paque PLUS or Histopaque-1077. Fourth, centrifuge at 400 x g for 35 minutes at room temperature. During this step, the granulocytes, platelets and RBCs pellet to the bottom of the tube and the PBMC float over the Ficoll-Paque PLUS or Histopaque-1077. Fifth, carefully aspirate the PBMC from the Ficoll-plasma interface. Sixth, wash the PBMC with PBS at 300 x g at 4° C twice. Thus the protein of your interest SHOULD have minimal RBC contamination.

Best wishes,

Ilya

Thanks Ilya. Next time onwards I shall lyse the RBC. The present sample turned mud color on heating and is turbid now. Any chance of retreveing them?

Thanks

Namita

Simply lyse by using ACK lysis buffer and collect the  solution after centrifugation by excluding WBC pellet.

--- Best Regards, 

Babu