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About ridhima

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    Molecular biology
  1. I want to know in pET28a/BL21DE3 and also in pCold TF/BL21DE3 for how much time (hrs) after induction with IPTG culture should be kept and at which temperature so that soluble aggregates should not form? I kept at 18 degree for 18 hrs after induction in pET28a and at 15 degree for 24 hrs but there is formation of soluble aggregates in both as detected by Native PAGE . I read that if kept for long time protein forms aggregates or come in inclusion bodies.
  2. Thanks Sir for your solution. Now I am clear. I have added DTT in buffer used for gel filtration that's why protein's molecular weight from SDS-PAGE and gel filtration is approx. 40 kDa and from Native Page it is coming approx. 146 kDa i.e. actually it is homotetramer.
  3. My protein is eluting at 80 ml in gel filtration. How can we say it is tetramer or monomer and how are you comparing this with SDS-PAGE results? Please explain to me in detail?
  4. My protein molecular weight is coming 40.7 kDa by gel-filtration and by gradient native page it is coming near 146 kDa and by SDS-PAGE it is coming near 40 kDa. I am not understanding this result as by gel- filtration and native PAGE molecular weight should come approximately same.
  5. My question is that when eluting protein with elution buffer we set pH of elution buffer (sodium phosphate buffer) to be 7.5 but after adding 500mM imidazole pH of buffer increases to 9. I want to know is there need to adjust pH of the elution buffer after adding imidazole to 7.5. If yes, then how can we set the pH by using NaOH or HCl or by adjusting with sodium monobasic or dibasic buffer salts or make buffer of less pH like pH 6.0 and after adding imidazole the pH will reach 7.5 after doing the standardizations that how much pH buffer to make so that after addition of imidazole it reaches 7
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