I often used HisTrap HP (Ni Sepharose) as the 1st column in my purifications. However, it pays to ensure that your soluble fractions is very, very clean (no insoluble protein) before loading onto the column. Centrifuge the heck out of it.

Even with very clean sample it can still take a while to load these columns (I have more experience with the 1ml format). Thankfully, the washing and elution steps usually go faster. I've run these columns as slow as 0.5ml/min, and I think even lower during the loading step. You may have to deal with the long loading time.

If you're impatient, or you need to load a ridiculously high volume you can instead perform the loading step with a peristaltic pump jury-rigged to the column and set at a low flow rate... I'm not a fan of this method since it risks 'destroying' the column since there is no pressure monitoring. Worse, you need to make sure the flow rate and volumes are known, since you'd need to let this sort of thing run all day or overnight, and you don't want to accidentally load air into the column if you oversleep or hit traffic.

The risk of destroying the column is higher with your column as well, since Ni NTA is agarose rather than sepaharose based. While the materials are functionally equivalent in this application, sepharose seems to tolerate abuse a bit better than agarose. That said, many FPLC columns can be recovered unless they are very, very seriously abused (not true for sizing columns).

For what it’s worth, there are fast-flow (FF) format Nickle columns, and a format called FF Crude that can allegedly handle some very dirty samples (linked). I never tested them, but I wanted to, for the specific reason that sample loading was taking so long (also the fast flow columns can take more abuse). I mostly used GE products since I was using a GE FPLC system (Akta), but it’s often possible to improvise systems to use other manufacturer’s columns.

Another possibility include a beginner’s mistake: NiNTA is usually stored in 20% ethanol. You'll need to wash the ethanol out of the column with water, then equilibrate the column with your buffer before loading the sample. If you accidentally loaded buffer or your sample to an unprepared column either the salts in the buffer or the proteins could precipitate and clog the column. If that happened you may need to thoroughly clean and possibly re-condition the column (see manufacturers instructions) -- if you cannot get it running again by following the previous post's suggestion.

http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-us/products/AlternativeProductStructure_17555/11000458

http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-us/17525501

Try to connect the male and female nut of the machine together without your column and run it with 2ml/min flow. If it gives the same alarm it means that the machine tubing itself is blocked and you can follow the tubing piece by piece until you find the blocked part. However, if it doesn't show any alarm it means that you column is almost blocked. In case if your column is blocked, try to run the column upside down for a few minutes with just buffer. It will probably clean it it up and hopefully resolve your problem.

May be try to filter your lysate by 0.2 mm filter before you load your lysate on Ni+2 column.

I often used HisTrap HP (Ni Sepharose) as the 1st column in my purifications. However, it pays to ensure that your soluble fractions is very, very clean (no insoluble protein) before loading onto the column. Centrifuge the heck out of it.

Even with very clean sample it can still take a while to load these columns (I have more experience with the 1ml format). Thankfully, the washing and elution steps usually go faster. I've run these columns as slow as 0.5ml/min, and I think even lower during the loading step. You may have to deal with the long loading time.

If you're impatient, or you need to load a ridiculously high volume you can instead perform the loading step with a peristaltic pump jury-rigged to the column and set at a low flow rate... I'm not a fan of this method since it risks 'destroying' the column since there is no pressure monitoring. Worse, you need to make sure the flow rate and volumes are known, since you'd need to let this sort of thing run all day or overnight, and you don't want to accidentally load air into the column if you oversleep or hit traffic.

The risk of destroying the column is higher with your column as well, since Ni NTA is agarose rather than sepaharose based. While the materials are functionally equivalent in this application, sepharose seems to tolerate abuse a bit better than agarose. That said, many FPLC columns can be recovered unless they are very, very seriously abused (not true for sizing columns).

For what it’s worth, there are fast-flow (FF) format Nickle columns, and a format called FF Crude that can allegedly handle some very dirty samples (linked). I never tested them, but I wanted to, for the specific reason that sample loading was taking so long (also the fast flow columns can take more abuse). I mostly used GE products since I was using a GE FPLC system (Akta), but it’s often possible to improvise systems to use other manufacturer’s columns.

Another possibility include a beginner’s mistake: NiNTA is usually stored in 20% ethanol. You'll need to wash the ethanol out of the column with water, then equilibrate the column with your buffer before loading the sample. If you accidentally loaded buffer or your sample to an unprepared column either the salts in the buffer or the proteins could precipitate and clog the column. If that happened you may need to thoroughly clean and possibly re-condition the column (see manufacturers instructions) -- if you cannot get it running again by following the previous post's suggestion.

http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences-us/products/AlternativeProductStructure_17555/11000458

http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences-us/17525501

Hello Liyana

I agree with Soran. First, you must be sure that your system is not blocked in somewhere. Remember that in chromatographic procedures the filtered of samples and solutions before applying into the column is mandatory. Samples must be soluble completely and stable in  binding and elution buffers. On the other hand, I have never worked with a Ni NTA column of this firm, therefore I am not sure that you are using the correct flow and back pressure. Be sure that your running conditions are the suggested by the manufacturer.

good luck!!!

Hi Liyana,

apart from the other good suggestions, what do you see: Is the pressure during equilibration already high? Or is the pressure increasing during the loading of your sample onto the column. If the first one is the case, check whether your plunger compresses the resin which leads to an increase of the pressure. Another reason could be a clogged filter above the resin which you could exchange.

If the second is the case then you load to much of your sample onto the column which which also increases the pressure.

And as already mentioned before: Always centrifuge your sample heavily before you load it onto the column! At least. I even recommend to filtrate the samples through a 0.2 µm filter.

As people have said, check your system pressure, then "column on" pressure.

Your system pressure should be negligible.

Some good tips for your initial capture are:

During lysis:

Either add Benzonase and/or continue the lysis until the DNA has been chopped up reducing the viscosity of the lysate.

After lysys:

A good hard spin, try to pipette off rather than decant as you can often pour off insoluble material and get a suspension.

If your lysate is still viscous dilute with binding buffer.

Your column material, is it the right material for your application?

The Generon HiFLiQ is 40 um beads, which is similar to the HisTrap HP 34 um resin, which is great for high resolution  small volume purifications using gradient elutions, but often not suitable for more general use. If you do step elutions you don't need a small bead column.

For very crude preps use the HisTrap FF Crude (as mentioned above), which has a 20um frit for better flow rates and uses FF beads which are 90 um beads, The greater interstitial space allows better flow or HisTrap FF can be used too. We use these columns a lot for High Throught put purifications for crystallisation for multi-milligram quantities.

Always follow the pressure rating of the resin 0.5 Mpa, if you run at too high a pressure the beads deform and the interstitial spaces collapse which effectively begins blocking the column.

For column recovery post  purification, wash very slowly backwards. You want to remove any particles and insoluble material from the column rather than trying to push it through. Once you done an initial wash backwards then flow in regular directions for CIP. If you have done a particularly large volume purification it may be necessary to incubate with 0.2 or 0.5 M NaOH overnight at RT

+1 to benzonase, or DNAse/RNAse treatment in general. Forgot to mention that.

Try to filtrate your protein sample by 0.2 μm filter connected with 0.45 μm filter before sample loading.

And, I recommend you that the crude sample obtained by ultrasonication and/or disruption of host is cooled on ice for 0.5 to 1 hour before centrifugation. 

AKTAs have a flow restrictor downstream of the HiFlIQ. The unit code is FR-902. First, disconnect the flow restrictor from the AKTA to see if the leak has stopped. If the leaking has stopped, you have 2 options. Either run the AKTA without the flow restrictor (which is not absolutely necessary) or pull the restrictor apart (with your hands) and adjust the back pressure with a Philips screwdriver. It's a very easy task.