Hi everyone,
I have expressed 6x histidine tagged protein(35kDa) and purified using Ni-NTA agarose (Qiagen). The elution buffer contain 20mM of Sodium phosphate, 500mM NaCl and 250mM Imidazole. I concentrated the eluted protein using amicon ultra centrifuge filter. 8 tubes of 500uL of eluted protein was concentrated and washed 5 times with 1xPBS pH 7.4 (500uL each). I measure the concentration of protein, the 260/280 ration was high which was 1.05 and 1.86. I used the purified protein to run ELISA and there was no signal at all (same as blank).
Any suggestions to improve the purity of protein (260/280) because I concern that imidazole is still there and effect the ELISA results.
Here are the couple of things that can be considered;
Could it be something derived from ELISA application instead of efficient protein purification? Please provide some info about ELISA setup...
Does the selected primary antibody in ELISA target the His-tags as antigen and if yes does the tag on protein reachable by antibody?
Is it possible to be occur a non-specific binding loss during ELISA assay?
Did you confirm your expression and/or purification by PAGE and Western?
Other than direct protein absorption measurement, protein specific assays e.g BCA, Bradford would be more reliable to assess...
To cleanup;
You may follow imidazole by its characteristic absorption, it absorps UV nearly at 210 nm...You may estimate the residual concentration (roughly)
You may decrease the NaCl conc if applicable for you...
Have you ever tried dialysis instead of Amicon?
What was the MWCO of the ultrafiltration membrane?
You may precipitate the POI if the concentration is enough (TCA, AS, acetone, phenol, etc...)?
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