I once tried HEPES lysis buffer for tissue lysis to obtain protein. After homogenization and sonication , i centrifused and collected the supernatant for protein as per the protocol. but later on again pellet was formed. why is this pellet again and again in supernatant too? . In order to confirm wht is this pellet . I ran a gel for B-ACTIN consisting on supernatant , that pellet solution and mixture of both in each well.. In that i got nice band for supernatant but a less intence band in the pellet solution .. so its clear that supernatant should be considered for for western . the main question is why is that pellet is forming again and again after centrifugation? .. Now i am using 8M urea buffer and all works fine.. is there any specific reasons for the use of urea buffer for western blotting ???? Is the use of urea buffer can hamper the quality of band in western blotting? Thank you in advance.
You said, "...but later on again pellet was formed".
You need to tell us when you see the supernatant forming pellets. Is it after a freeze-thaw cycle or without any freeze-thaw cycle? If without any freeze-thaw cycle, where do you store the lysate between two centrifugations, and how long, and why? It is perfectly normal for a whole cell extract (WCE) preparation to precipitate to some extent after each freeze-thaw cycle; even this happens with reasonably cleaner preps like a nuclear extract (NE). This is because several proteins lose their folding architecture upon changes in temperature, and many such proteins are differently susceptible to "salting-out" by the salt and other components in the buffer. This is why it is advisable, always, to aliquot the lysates/extracts in small portions and to avoid repeated freeze-thaw cycles.
Your observation may also be due to a faulty buffer. But, you have not told us the composition of your "HEPES lysis buffer"; there is no standard lysis buffer of that name so that people would understand what it is. People can make many varieties of lysis buffers with Tris, Hepes or any other buffering components and variously use the buffers to lyse a variety of cell/tissue types and nuclei. Based on different cell/tissue compositional architecture the many lysis buffers differ in composition.
To be able to assess if your buffer has any flaw, you also need to tell whether you purchased the lysis buffer from a company (tell us the cat no., if so) or you prepared it yourself. In that case, you need also to tell us how you made the buffer (from what stock solutions, and how you prepared the stock solutions).
Most standard cell/tissue lysis protocols avoid use of urea if the downstream operation involves SDS-PAGE; but exceptions exist. This is because proteins gain chemical carbamylation when heated in presence of urea; this impairs protein mobility on gel and also interferes with mass-spectrometric procedures. Urea is a strong chaotropic agent and helps denature the proteins, and it has its own specific usages. Whether it is best for you to use urea in your protocol is a question you should address after some studies.
theoretically urea should not affect transfer because it is uncharged and it solubilized some large and hydrophobic protein. you may be increase your RPM in centrifugation so this might be helpful to get clear supernatent
@anil panigrahi sir ... Thank you sir for your feedback and suggestion in your busy schedule. Ya i observed pellet formation after freeze thaw cycle.. thank you for making my concepts more clear. The HEPES lysis buffer which i used for brain tissue samples consists of freshly prepared stock solution of 1M HEPES, 1MMgCl2, 0.5M KCl, 0.1MPMSF, 0.1M DTT, 100 xPI from this i prepared around 5 ml and volume was made with ultra pure water. after preparation of this buffer, solution was clear.
Thank you sir once again.
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