Can I connect the column to a peristaltic pump operated at a low flow rate and leave the other end open to elute the contaminants out of the column? Or will it disrupt the column packing??
Hi. Yes, you can use a peristaltic pump, as long as you respect the flow rate suggested by the manufacturer. Calibrate your pump before connecting it to the column and you should be fine. Watch for bubbles.
Hi. Yes, you can use a peristaltic pump, as long as you respect the flow rate suggested by the manufacturer. Calibrate your pump before connecting it to the column and you should be fine. Watch for bubbles.
Dear Sundarya
As Patric told, you can certainly do it.
You can use peristaltic pump for all low pressure columns as 16/60 and 26/60 Sephacryl and Supedex. Of course you need to pay attention to the bubble and as Patrick told verify the effective flow that you pump provide once that the columns it is connected, because the counter-pressure can reduce the real flow.
You can simply set a theoretically flow, measure the real flow by measuring the volume collected at the out of the columns in 10 minutes and do this until you found the theoretical flow that guarantee a real flow close to the one suggested for the columns from GE.
Several years ago before acquire the first AKTA, I setted-up a simple manual system using:
A GE P1 peristaltic pump connected with an Injection valve (manual – 6 port 2 position – 1 port for the siringe, 2 port for the loop, 1 port for the column, 2 port for the waste - https://kinesis-usa.com/rheodyne-idex-health-science-medium-pressure-valves-medium-pressure-injection-valve-6-port-2-position-bulkhead-manual-v-451.html) that allow to perform sample injection manually, connected with the coloumn which was also connected with a Frac-920 fraction collector which was manually started at the moment of sample injection.
This simple and cheap system was be used several times to perform semi preparative SEC purification that do not require gradients. Its work very well. The main drawback was related to the absence of the 280UV detector and therefore you have to run in the SDS page more or less all the fraction that you collect.
A possible solution to decrease the fraction number to be loaded is perform a fa rapid 96 well colorimetric assay with Bradford reagent 1X (eg. 100ul od Bradford reagent 1X + 10ul of protein from a single fraction) and check which fraction provide to you a blue coloration that indicate protein presence. However, you can do it, if you work with concentrated proteins that you are sure that provide a good signal with Bradford, because coupling the low sensitivity of Bradford with the protein dilution (5-10 times) that may happen during the run, you can risk to do not consider fraction that contains the protein but are negative to the Bradford. Therefore also if you reduce the fraction number to be loaded in the gel on the basis of the colorimetric assay result, do not through away the other fractions until you do not see the SDS-page ans you are sure that your protein is present in the loaded fractions
Sorry for the long message.
Good luck
Manuele Martinelli
p.s: if you are interested I’m building a blog (http://proteocool.blogspot.com/) with some suggestions regarding protein production and purification.
Thank you for your inputs Manuele Martinelli and Patrick Jack Spencer
Yes. Use 0.1-0.2 ml/min of flow rate. The pressure will not increases and the column will work properly.
Yes, if the flow rate 0.5 ml/min, or lower. The max flow rate for that column is 1.0 ml/min.
https://www.gelifesciences.com/en/us/shop/chromatography/prepacked-columns/size-exclusion/hiprep-sephacryl-s-100-hr-p-05799#tech-spec-table
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