I want to isolate secretory VacA protein from a broth culture. After salting out, I'm performing dialysis with 1X PBS (pH 7.5) and passing 1 ml of sample through self made 2 ml Sepharose 6B column at 4 oC. I'm using same 1X PBS (pH 7.5) for elution. I'm collecting fractions for one minute each and after SDS-PAGE only getting same pattern of protein bands in all fractions without any single band of 88 kDa. Now, I'm wondering whether it is possible to isolation a single protein by a self made gel permeation chromatography column of 2 ml having height of
Hi Sandeep, gel filtration is not generally used as a primary chromatography method to isolate a protein from a crude mixture.
You should use cationic/anionic/hydrophobic resins for initial purification like Ashwini suggested.
Dear Sandeep Kumar,
Could you tell me what is the amount of total protein being loaded in the sepharose. column? What I feel is that you are loading a higher quantity of sample to your sepharose GPC column which is not allowing you to get your proteins being separated with good resolution. And you are getting a similar pattern of bands in PAGE.
You can load about 25 mg of your crude protein sample and subsequently collect and check the purity via SDS-PAGE. If it again gives you the same problem then you need to load further lower amount (say 10-12 mg).
You can load about 25 mg of your crude protein sample and subsequently collect and check the purity via SDS-PAGE. If it again gives you the same problem then you need to load further lower amount (say 10-12 mg).
Moreover, you must try cation or anion exchange column as well to refine your separation. You can try C18 sep-pak cartridges (tiny hydrophobic interaction chromatography column) too.
A combination of Gel filtration, Ion exchange and hydrophobic interaction chromatography will help you get your almost purified protein (>98%).
Regards,
A K Dubey
Gel filtration is limited by the amount of sample you can inject. For fractionation inject a sample volume equivalent to 0.1% of your column volume. If you want to purify larger amounts of protein then do not use gel filtration since you will require a gigantic column.
Hi Sandeep, gel filtration is not generally used as a primary chromatography method to isolate a protein from a crude mixture.
You should use cationic/anionic/hydrophobic resins for initial purification like Ashwini suggested.
I agree with the preceding comments. If I have a 1 mL sample of fairly concentrated protein, I'll typically use a 1.5 cm diameter Bio-Gel or Sepharose column at least one meter in length, and even that is too small at times. Your 2 mL column is much too small to accomplish any significant separation of a 1 mL sample. That's why people usually use a high-capacity method like ion-exchange or hydrophobic interaction chromatography as one of the first steps, and only use GPC as a final "polishing" step.
Thank to all, these are some valuable suggestions which will definitely help me in my future experiments
I agree with Steingrimur - I would add an affinity column if you have an antibody specific for your rProtein.
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