The mol wt of my protein of interest is about 27 Kda but i am getting the signal at approx 50Kda. I am Using polyclonal antibody and the standard 12% gel for the detection. Running gel conditions using BIO RAD assembly is initially at 70V (Stacking) and 150V (Resolving). Transfer is by semi-dry method (using BIO RAD Turbo Blot) followed by blocking in 5% milk prepared in 1X PBST (0.1% Tween 20) for about 4-6 hrs or sometimes overnight (since my antibody comes up with numerous black dot on whole of the membranee if i go for 1 hr blocking) followed by pri antibody at 4C overnight or 2 hrs at RT and 2 hrs at 4C followed by washings with iX PBST for 30 minutes at an interval of 5-10 minutes. Then incubation with sec antibody for 1.5 hrs at RT prepared in 3% milk (in IX PBST) and again washings as mentioned above and then detection. Pri Ab dilution is 1:500; Sec Ab dilution is 1:20000. Somwtimes the antibody won't work at all and at other times it gives signal at around 32Kda. An image has been attached as well. Please suggest some troubleshooting so that i can get band at around 30Kda if not at exact 27 Kda and why would signal be strong at 50Kda. Thanks
Your blot looks like the antibody is unspecifically binding to all proteins. Perhaps the antibody concentration (either of the primary and/or secondary) is too high. For the secondary antibody, follow manufacturer's instruction; for the primary you need to titer yourself.
Dimers? Should not happen with reduced SDS-Page, but I have had it happen to me with one protein.
It could also be some form of aggregation. How do you treat you protein before loading? If you heat it at ~95C, try lower temperatures, e.g., 60- to 80C.
If you use 2-mercaptoethanol and if the bottle is older than 4- 6 months, or if it was stored for a longer period at RT, use a fresh one. BME oxidizes very easily with air; or use DTT, which is stable.
Dear Vikrant,
there are proteins that form SDS-resistant oligomers. Your observed molecular weight might correspond to the size of a dimer. Is your protein likely to form a dimer?
Your observation might also be related to your SDS-gel sample preparation. Did you boil your sample in SDS buffer prior to loading and was the buffer still fresh (presence of reducing agent)?
Best,
Jan
Achim Recktenwald I heat my sample at 95C for approx 5 minutes before loading them. I can try o to lower the temperature and then see what happens.
Jan-Hendrik Heilers Yes the buffers have been freshly prepared. About the dimers i also thought that if there is some dimerisation but no the protein is a monomer of about 27 Kda.
Engelbert Buxbaum Yes may be i can try to dilute the antibody (Pri as well as sec) and then observe what happens
Most likely your antibody is not specific for our target (e.g. see BAKER M., p. 274, NATURE. VOL 521 Date: 21 MAY 2015.). Alternatively, uncertainties could be reduced via revising the protocol, used chemical, tools, apparatures, methods as well as experimentator's work.
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