Hello.
I have purified my protein with Ni-NTA coulmn and i have run my purified fractions on SDS-PAGE but i don't know how to calculate the purity of may bands. how should i do this?
Thanks in advance.
You can download ImageJ software (https://imagej.nih.gov/ij/). Using this software, you can select individual lanes on your gel and then get a color intensity profile across the lane (you'll want to convert your image to black and white probably first). You can then integrate your protein peak area and divide by the sums of the peak areas for all of the bands in the lane. Here's a Youtube video that details how to select lanes (they only select a single peak, but the principle is the same) and integrate peak areas: https://www.youtube.com/watch?v=JlR5v-DsTds
You can download ImageJ software (https://imagej.nih.gov/ij/). Using this software, you can select individual lanes on your gel and then get a color intensity profile across the lane (you'll want to convert your image to black and white probably first). You can then integrate your protein peak area and divide by the sums of the peak areas for all of the bands in the lane. Here's a Youtube video that details how to select lanes (they only select a single peak, but the principle is the same) and integrate peak areas: https://www.youtube.com/watch?v=JlR5v-DsTds
To calculate an absolute protein amount you have to use an internal sample standard with a known concentration. The Smart Protein Layers technology for 1D gels could be helpful there as there is a specific standard in the sample buffer. With this you can calculate the absolute protein amounts of your protein bands using ImageJ or LabImage. This way you will get absolute amounts not only relative ones.
Hi,
to calculate an absolute protein amount you have to use an internal sample standard with a known concentration. The Smart Protein Layers technology for 1D gels could be helpful there as there is a specific standard in the sample buffer. With this you can calculate the absolute protein amounts of your protein bands using ImageJ or LabImage. This way you will get absolute amounts not only relative ones.
This technology is fluorescence based as fluorescence has a much higher dynamic range. with Coomassie or silver quantification is not possible as their linear range is very low. You come into saturation very fast (not seeing it really) so you can not determine the protein amount by band intensity values anymore!
If the protein of interest has a heme group, ie it is a hemeprotein, you could check the purity ratio through an electronic spectrum in the ultraviolet and Visible region, making a ratio between the absorbances at 280 nm and the soret band.
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