I am purifying a his-tagged protein. My vector has the his tag and a V5 epitope. I am using anti-V5 antibody for my western. On the western blot, I see a band at the correct molecular weight, but I also see band at almost twice and thrice its size. Can anybody explain why?
Sounds like you do not have enough reducing agent in the SDS/loading buffer and the protein is making dimers/trimers via disulfide bonds. Try adding fresh DTT and heat to 95oC.
Dear Amruta, perhaps you need to check that your purified protein makes dimer or trimer along with monomer..
Could you attach the gel picture..in most case it would be a non-specific band. you can try a negative control where the protein of interest is absent. Do not decide the oligomeric state of the protein with this.
Sounds like you do not have enough reducing agent in the SDS/loading buffer and the protein is making dimers/trimers via disulfide bonds. Try adding fresh DTT and heat to 95oC.
Thanks everyone. Gary, I am running a new blot today with some fresh sds. I hope it works. Thanks for the advice.
Gary, how long do I heat at 95oC? I generally heat my samples at 95oC for 5 minutes. Is it enough. I get different answers from different sources.
5 min. is plenty, again make sure you have plenty of fresh DTT in there.
Add DTT and make sure you don't overload. High protein conc. tends to form multimers on SDS even if DTT is fresh and plenty because DTT is removed quickly from the sample in the gel matrix.
If unsure, add DTT/BME to running buffer and pre-run gel 30 min before loading sample so the gel is saturated with reducing agents.
Hi Daniele,
I am going to do SEC analysis and then try to check whether the sizes match up using proteomic approach.
Thanks for the advice.
Overloading of proteins in the gel might cause such problems. Befor loadingvthem you would better to do a BCA asay to load appropriat amount of protein into the gel.
Hi Gary, I try to answer, in few words, your proper request of clarification about SEC and MALLS.
Analytical Size Exclusion Chromatography allows to define the apparent MW of protein contents of your sample. The presence of aggregates (huge problem if not removed from sample) or oligomerizzation status of Protein of Interest. While Preparative SEC allows separate you protein from others forms, contaminants and aggregates. Multi Angle Laser Light Scattering in line with SEC, define the correct MW of the protein species which are present in your sample.
Try out beta mercaptoethanol in your loading dye...prepare before use..Thick band you are getting because of dimer or trimer..so you have to denature it properly..boil it for long time and vortex it after every 5 mins of boiling..
all the best.
Daniele,
Thanks, figured out SEC (I just never use that abbreviation), and MALLS was a new abbreviation for me, have had light scattering used to analyze my samples, but never in line with SEC.
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