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TOPIC: Experimental problems in amino acid - protein interaction study?

Experimental problems in amino acid - protein interaction study? 16 Sep 2019 16:02 #1

  • Charlesmsa7
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Hello Arnoud,
I will try to elaborate the experiment in details. Kindly go through and suggest possible solutions for this problems.
Protein is of 18 kDa (exactly 17.9 kDa), calculated PI value is 8.4. Parent protein solution (5mg/ml) was in Buffer containing Tris - 50mM, EDTA - 0.04 mM, MgCl - 0.1mM, Tween20- 0.05mM, DTT - 0.1mM and HCl- for pH adjustment.

#1.
2 microlitter form parent solution was diluted in acetate buffer (pH 5.5, from GE) and made to 200 microlitter. This solution used for immobilization by passing for 15 mins at 10 ul/min flow rate on CM5 chip using amine coupling. Overall only 33.9 RU only achieved. What could be the possible reason for very lower immobilization rate?.

#2.
5 amino acids (cysteine, proline, tryptophan, lisyne and histidine) were dissolved in running buffer contains Tris - 50mM, EDTA - 0.04 mM, MgCl - 0.1mM, Tween20- 0.05mM, DTT - 0.1mM and HCl- for pH.
Five concentrations of amino acids were injected (low to high) as such as 0 (3 cycles), 31.25 (1 cyc), 62.5(1 cyc), 125(1 cyc), 250(1 cyc), 500 (1 cyc) micro moles.
Assay performed using kinetics concentration series program with the following parametres.
For Reactions: flow rate 60 ul/min, stablisation 0, injection 1 min, dissociation 3 min,
For regeneration: regeneration using running buffer, flow rate 60 ul, for 3 mins,

The problem here is i could not able to get response. Except cysteine, for others the reaction peak goes negative. For lysine and tryptophan only the highest concentration shows positive peak.
How can I optimize the experiment?

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Experimental problems in amino acid - protein interaction study? 17 Sep 2019 19:50 #2

#1: Suggestion: look at www.sprpages.nl/immobilization/ligand-pre-concentration .
Second, the buffer of your protein is not favourable for immobilization. The TRIS will interfere with the amine coupling. The concentration of the protein is 50 ug/ml during immobilization. Did you observe any analyte pre-concentration?

#2: When the immobilization is very low as in your case you will not find any response since there is not enough protein to react with.
You can calculate the Rmax of the reaction with the formulas at www.sprpages.nl/immobilization/immobilization-procedures .
In addition look at your other question ( www.sprpages.nl/kunena/new-forum/560-exp...protein-interactions ).

As a remark: it is better to inject the concentrations ad random and interspersed with some blanks to avoid systematic errors.

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