I have 1 ml of 0.4 mg protein which I want to separate. Mainly I want the complex which should come in the void volume. I used a 40 ml bed volume manually packed column (0.75 cm diameter), but didn't get any protein back. Could you please suggest?
Sephadex G200 is not a great choice for this separation because the 100,000 Da protein will not be well-separated from the larger complex. Both should have come out early, but since you got no protein out, they must have stuck to the column. If the tubulin polymerizes into microtubules, it will probably get stuck at the top of the column.
If you are using manual chromatography, you might try Bio-Gel A-0.5m.
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006139.pdf
Bio-Gel is made of a different material than Sephadex, so your protein may not stick to it as much. You may find it useful to include some salt (0.3 M NaCl) in the elution buffer to minimize electrostatic interactions with the resin.
For Sephacryl, you would probably use S200HR or S300HR.
For gamma-tubulin complex separation, would sephacryl be better than sephadex?
Sephadex G200 is not a great choice for this separation because the 100,000 Da protein will not be well-separated from the larger complex. Both should have come out early, but since you got no protein out, they must have stuck to the column. If the tubulin polymerizes into microtubules, it will probably get stuck at the top of the column.
If you are using manual chromatography, you might try Bio-Gel A-0.5m.
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006139.pdf
Bio-Gel is made of a different material than Sephadex, so your protein may not stick to it as much. You may find it useful to include some salt (0.3 M NaCl) in the elution buffer to minimize electrostatic interactions with the resin.
For Sephacryl, you would probably use S200HR or S300HR.
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