Hi all,
When I do the exact same IP protocol, but elute with different buffers (for example, in 50 µl LDS or LDS together with sample reducing agent and MQ water, or in 1X Laemli), I get different bands on Western blot. What does this mean?
Kind regards,
Stijn
Well you should give more details on your experiment but straight away any different elution buffer might give you different results yes that's normal. Between reduced and non reducing conditions, strong or mild detergent the results may greatly vary
Don't you think elution is the most critical step of this experiment? If so, while trying to vary the composition of the elution buffer it is very much obvious that the result will end up differently. Probably, the affinity of the protein of interest is getting affected here, on altering the elution buffer. l suggest sticking on to a single protocol.
Thank you all for your suggestions.
See the blot below:
- In lane 1 & 3, you can see the eluate of the same immunoprecipitation experiment, but in lane 1 it is eluted in 50µl LDS (after which 5µl of this eluate was added to 13µl MQ and 2µl sample reducing agent), while in lane 3 it was immediately eluted in 32.5 µl MQ, 12.5µl LDS and 5µl SRA.
- Same as for lane 2 & 4, where a different immunoprecipitation experiment is eluted with the same as 1 & 3.
As you can see, with a different elution, I lose a band.
Would elution be so crucial that it is due to the elution that I never detect my protein of interest?
It clearly looks way different and it's probably due to the reducing condition. In lane 1 and 3 you use a non reducing elution which might give you still associated proteins or multimeric forms which are not totally resolved during the migration.
Lane 2 and 4 you are eluting with reducing agent which is separating proteins which are usually associated.
That could be the difference, you are probing your blot with a specific antibody ? Does the strong band in lane 3 and 4 fit with the predicted size of your target protein ?
- Badges
- Science method