I have been looking into the literature and different protocols for some answers pertaing to the stability of western blot phospho-protein samples in lysis buffer, in loading buffer, and after transfer onto the PVDF membrane. My questions are; how stable are western blot phospho-protein samples in lysis buffer at -20 C, 4 C, and -80 C? How stable are these samples once constituted in loading dye? Can western blot samples be constituted in loading dye and saved at -20 C? if so, for how long? How stable are the phospho-protein samples after transfer to PVDF membranes (Given that samples are always kept at -20 C, and only thawed on ice or at 4 C for constitution and analysis, and the membranes are always frozen if they are not being probed or reprobed for another protein) ?
In lysis buffer plus phosphatase inhibitors on ice they are not very stable and should be transferred to the protein loading buffer as fast as possible due to the activity of the phosphatases, in loading buffer they should be stable at -20 degrees.
They should be very stable...I normally keep them dry, if I am using pvdf. The bigger issue is in your sample buffer.
The collegues are right in that on a dry PVDF membrane phosphorylated aminoacid side chains should be stable. However, we made the observation that phospho-specific antibody Western blots worked best on freshly blotted and blocked membranes. I do not know why this is so but this is what we saw with phospho-Serin-133-CREB, pERK1/2, pAkt, p38 and many others. Absolutely deleterious for amino-acid-phosphates is India Ink staining, since this ink binds organic acids and amino-acid phosphates.
In lysis buffer plus phosphatase inhibitors on ice they are not very stable and should be transferred to the protein loading buffer as fast as possible due to the activity of the phosphatases, in loading buffer they should be stable at -20 degrees.
Nicole Paland is absolutely right, wherever possible we put potential phosphoprotein-rich samples directly into 2 x SDS (or LDS) loading buffer. In that state (and at -20 degrees C) they last years. When thawing these sample repeatedly, however gels sometimes show a tendency to smear and DTT or mercaptoethanol should be replaced and the denaturing protocol repeated.
I have found phospho proteins have highly variable stability. I would recommend working with them quickly and transferring to membrane to store. During western blots be sure to add phosphatase inhibitors into your antibody dilutions, NaF 20 mM maybe even others. They are usually good for reprobing once in this state. I store samples at -80 in lysis buffer and try to keep freeze thaw to an absolute minimum. My interactions are phospho dependent and show high variability so I tend to be a bit picky with this sort of work. Perhaps a bit overkill but it works!
I can also say with confidence that the phospho residues remain stable when stored below freezing (however, I always keep my lysates in generic lysis buffer spiked with phosphatase inhibitor cocktails from Sigma and DTT/BME at -80). Depending on the protein/phosphorylation, you may consider aliquoting to prevent multiple freeze-thaws. Another consideration is to be sure that you have the complete spectrum of phosphatase inhibitors too if you are sourcing from Sigma and are unsure about what kind of residue phosphorylation you will be looking at.
I've never had a problem with stability after transfer on blots, but I also stain immediately on freshly transferred blots. I had the best experience using the fast, dry blotting iBlot system from Invitrogen.
Is a buffer of this constitution (137 mM NaCl, 20 mM TRIS, 1 % NP40, 10 % glycerol, 1mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml aprotinin, 1μg/ml leupeptin, 0.5mM sodium) convinient as phospho protein lysis buffer??
(Am sorry to bring in another question rather than an answer)
As long as you have phosphatase inhibitors (cocktail) in your protein lysates, and your samples are stored in aliquots at -20 or -80, you are good and should have no problem. Having said this, if you are looking at a particular phosphoprotein, then this issue of stability would further depend on the inherent nature of this protein in question.
There should be phosphatase and proteinase inhibitors in lysis buffer. Samples can be stored at -20 for no more than 1 month. If you want to store longer, you should store in -80.
As Erik said I also use 2X sample buffer and it seems OK the only problem is that some times I get smear band for some of my samples...does any body have any idea why this happens and how can I recover those samples?
Smears band can also occur due to loss of DTT/mercaptoethanol and the resulting pattial renaturation. Addition of fresh agent (DTT) may help.
Thank you all fellows for your valuable feedback. That was a great help.
Millipore protein handbook states DTT inactivated sodium vanadate ? If this is true, be carefull for your phosphor tyrosine analysis
http://www.millipore.com/userguides/tech1/mcproto444
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