Sajid, As Antonio said, we need more information in order to really help you. Particularly what columns you used and a little more about the protein target. As one who has worked on mammalian tissues, I found that some proteins are very sensitive to proteolysis after homogenization, but others are like a rock. Also, are you sure that the protein band you saw on SDS-PAGE is your protein target? Do you have an antibody to detect your protein by western blot?
Michael P.S.: I know that we live in a more anonymous social society dominated by social media, but I believe that most scientists are globalists and welcome the open and free exchange of information. I wish that the younger users of this bulletin board would indicate where they are working (institute and department). Others on CCP4BB may know someone even next door who can help. RMG **************************************************************** R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334 Email: [email protected] **************************************************************** > On Jan 18, 2017, at 6:32 AM, sajid akthar > <[email protected]> wrote: > > Dear All > > I am purifying a protein from liver. In first step I spotted protein in Flow > Through and could see intense band in SDS PAGE. I pooled the fraction and did > second column. Surprisingly I could not see UV absorbance or even any band in > the SDS. I used PMSF as protease inhibitor from begining of purification. > First column I used Tris buffer (pH 8) and for second column I used MOPS > buffer (pH 6). > > What could be the reason? > > Thanks in advance > > Sajid
