I purify by FPLC by Hitrap SP FF column. I have 4 fractions and all of them were being collected. I take an SDS-PAGE electrophoresis, and get some things below:
- Percentage of target protein in Sample lane and sole lane which have target protein are 60%, 98% respectively.
- Flowthrough lane doesn't have target protein. This demonstrates that all target protein was bound onto column. They would be eluted in the next step.
- Wash lanes have a little other proteins but doesn't have target protein.
- 3 rest fractions don't have target protein.
- Performance of purification is 45%.
- After fractions, I wash column with NaCl 2M buffer to take column clean. It doesn't have any protein.
Something is wrong. All of the target protein was to be collected in one sole fraction but the performance is so low. Where is the 55% rest protein? Did FPLC destroy the protein structure to many oligopeptides?
it would be relatively rare for FPLC to degrade a protein so rapdily. which matrix did you use ? the stability of the protein would depend on the buffer conditions, and possible presence of proteases. was the column/ tubings contaminanted with fungal contaminants ? try washing the system with 0.1 M NaOH and 20% ethanol and else if the target is hydrophobic/ or the pH of the buffer is the isoelectric point of the protein it would have precipitated. try to elute the column with 20% acetonitrile. but you are also saying that you have a 98% pure protein in your elution fraction. why don't you scale up ?
Make sure of your calculations. How are you measuring the concentration of the protein of interest in each fraction? Are you multiplying that by the volume of the fraction? Are you taking into account that in an impure sample with many proteins, some proteins run at the same place on SDS-PAGE, so that the band you identify as your protein in the impure sample may actually contain more than one protein?
Hi there,
In case of on column aggregation/precipitation, some material might be stuck because 2M NaCl is not enough to disrupt interaction between protein and gel beads.
It might be due to aggregation of your target protein. There is a system filter in FPLC and a filter in the colum, if protein forms aggregation during injection, the aggregation could be arrested on one of those filters.
I still don't understand. If protein was be aggregated, the column or FPLC system would be obstructed.
Protein aggregates are not necessarily so large as to obstruct the flow through an FPLC column or system. Anything that large would be a precipitate. An aggregate may still be soluble. Loss of the aggregate on the column based on its size would require that the space between the resin beads was too small for the aggregate to pass through. This seems unlikely to occur with an FPLC resin. Another possibility is that the protein became denatured, precipitated, and became stuck in the column. If enough protein did this, the column would eventually clog.
@Much: I divide weight of target protein in eluted fraction by weight of target protein in sample.
@Adam: I don't now that how many case of protein-lossing are there exception of aggregation? Can you help me?
beside aggregation problem I would worry for the presence of active protease which might hydrolyse your protein. You might want to add protease inhibitor such as PMSF. The amount of protein in your sample before adding to the column should be high, since there is a possibility that during elution some protein may adhere to the column
@Maggy: Thanks. I'm used to use EDTA and PMSF to prevent protease. Both have and haven't protease inhibitor don't have difference.
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