Ni NTA Beads 6FF By Lablead biotech,

详情

Ni NTA Beads 6FF CAT：N****0 Price: $*2 5ml; $**3 *0ml ; $**2—*5ml; $**8—**0ml; $***9—**0ml; $*********0ml (negotiable)

product ID: N****0
Storage conditions: **8℃

Product introduction:
The Ni affinity chromatography medium produced by LABLEAD belongs to a class of metal chelating media, which is formed by using high flow rate agarose as matrix, subnitrotriacetic acid (NTA) or iminodiacetic acid (IDA) as ligand, and chelating metal ion Ni. The affinity medium not only has the advantages of large adsorption capacity, good selectivity, high resolution, ultra-low limit Ni2+ leakage, easy regeneration, and low cost, but also helps to maintain the biological activity of the product and improve the yield of the product, so that it is widely used in the separation and purification of downstream proteins, nucleic acids and peptides in biopharmaceuticals and bioengineering, especially the efficient preparation of histidine labeled proteins.
Ni-NTA 6FF beads Features:
Halogen can withstand harsh conditions such as a certain concentration of reducing agent, denaturant or coupling agent, has wider applicability, more stable ligand, higher selectivity, can withstand up to 0.3MPa pressure, is more stable, and can achieve purification of the target protein at a relatively high flow rate.
technical index：

Product name	Ni-NTA 6FF beads
Ligand	subnitrotriacetic acid (NTA)
Matrix	Highly crosslinked 4% agarose gel
capacity	*0mg his label protein
Microsphere size	*5%
maximum pressure	Soluble in DMF
pH stability range	*2（work），*4（washing）

1.Purification process
1.1Buffer Preparation
Buffer can use the following recommended Buffer, you can also configure different buffer liquid system according to your own use habits, the basic principle is low imidazole loading, high imidazole elution, or high pH loading, low pH elution. Buffer is best filtered with 0.*2μm or 0.*5μm filter before use. Because Ni NTA Beads 6FF can be used for the purification of soluble protein and inclusion body protein, the buffers required by the two methods are different. The specific configuration methods are shown in the following table:
Table 1：Buffer and formula for soluble histidine label protein purification:

Name	Volumn	formula
Lysis Buffer	1L	0 mM NaH2PO4（7.0 g NaH2PO4·2H2O）,*0 mM NaCl（7.4 g NaCl）,0 mM imidazole（0.8 g imidazole） Adjust pH to 8.0 with NaOH solution,Use 0.2 or 0.*5 μm filter membrane to remove bacteria.
Wash Buffer	1L	0 mM NaH2PO4（7.0 g NaH2PO4·2H2O）,*0 mM NaCl（7.4 g NaCl）,0 mM imidazole（1.6 g imidazole） Adjust pH to 8.0 with NaOH solution,Use 0.2 or 0.*5 μm filter membrane to remove bacteria.
Elution Buffer	1L	0 mM NaH2PO4（7.0 g NaH2PO4·2H2O）,*0 mM NaCl（7.4 g NaCl）,0 mM imidazole（7.0 g imidazole） Adjust pH to 8.0 with NaOH solution,Use 0.2 or 0.5 μm filter membrane to remove bacteria.

Table 2：Buffer and formula required for purification of inclusion body histidine label protein:

Name	volumn	formula
Lysis Buffer	1L	8 M Urea（*0.0 g urea）,*0 mM NaH2PO4（5.0 g NaH2PO4·2H2O）,0 mM Tris·HCl（5.6 g Tris·HCl） Adjust pH to 8.0 with NaOH solution,Use 0.2 or 0.*5 μm filter membrane to remove bacteria.
Wash Buffer	1L	8 M Urea（*0.0 g urea）,*0 mM NaH2PO4（5.0 g NaH2PO4·2H2O）,0 mM Tris·HCl（5.6 g Tris·HCl） Adjust pH to 8.0 with NaOH solution,Use 0.2 or 0.*5 μm filter membrane to remove bacteria.。
Elution Buffer	1L	8 M Urea（*0.0 g urea）,*0 mM NaH2PO4（5.0 g NaH2PO4·2H2O）,0 mM Tris·Cl（5.6 g Tris·Cl） Adjust pH to 8.0 with NaOH solution,Use 0.2 or 0.*5 μm filter membrane to remove bacteria.

1.2 Sample Preparation
1.2.1 Proteins expressed by bacteria or yeast
1. Single colony was selected into LB medium, and inducer with corresponding concentration was added for corresponding time according to carrier instructions.
2. After expression, the medium was transferred to a centrifugation cup for 7,**0rpm and centrifugation for *5min to collect the bacteria, and then 1/*0 volume of Lysis Buffer and PMSF were added before lysis with the final concentration of 1mM. Add lysozyme (working concentration is 0.**0.4mg/ml, if the expressed host cell contains pLysS or pLysE, you can not add lysozyme), (at the same time, other protease inhibitors can also be added, but can not affect the binding of the target protein to the resin).
3, the bacterial precipitate suspended, (if the concentration of bacterial solution is high, can also consider adding *0μg/ml RNase A and 5μg/ml DNase I), mixed, placed on the ice, and then the ice ultrasonic broken cells, until the bacterial solution is basically clarified.
4.Transfer the clarified crushing liquid to the centrifugal tube and centrifuge at *0,**0rpm for ****0 minutes at 4 degrees. Remove supernatant and set aside on ice or store at **0 ° C.

1.2.2 Yeast, insect and mammalian cells secrete and express soluble proteins.
1. Transfer the cell culture medium to the centrifuge cup, centrifuge at ***0rpm for *0min, collect the bacteria to obtain the supernatant, which does not contain EDTA, histidine and reducing agent, and can be directly added to the column for use; If it contains EDTA, histidine and reducing agents, it can only be added to the column by dialysis at 1×PBS at 4℃.
2. For a large volume of supernatant, it is necessary to add ammonia sulfate to precipitate and concentrate, and the protein can be added to the column after 1XPBS dialysis at 4℃.

1.2.3 Inclusion body protein purification (denaturation conditions)
1. Transfer the culture medium to a centrifuge cup at 7,**0rpm for *5min to collect the bacteria and remove the supernatant.
2. Lysis buffer=1:*0(W/V) is adopted to suspend the lysis, mix it well, and lysis is performed in an ice bath.
3. Transfer the crushing liquid to the centrifugal tube and centrifuge at *0,**0rpm for ****0 minutes at 4 degrees. Remove the supernatant, steps 2) and 3) can be repeated once.
4. The inclusion body is suspended from the Lysis :Lysis buffer(containing 8M urea)=1:*0(W/V).
5. Purify His label protein under denaturation condition. See Table 2 for specific buffer formula.

1.3 Loading of Ni NTA Beads 6FF
Ni NTA Beads 6FF is widely used in industrial purification, so it involves the filling of various medium pressure chromatographic columns. The following describes the method of filling chromatographic columns using Ni NTA Beads 6FF.
Loading of chromatographic column (using reservoir loading)
1. Rinse the sieve plate and joint at the bottom of the chromatography column with deionized water to ensure that there are no bubbles on the sieve plate at the bottom of the column, close the outlet at the bottom of the column, and leave ***0px of deionized water at the bottom of the column.
2. Suspend the resin and carefully pour the slurry continuously into the chromatographic column. A glass rod is used to pour slurry along the wall of the column to reduce the formation of air bubbles.
3. If a reservoir is used, the chromatographic column and reservoir should be filled with water immediately, and the sampling dispenser should be placed on the surface of the slurry and connected to the pump to avoid bubbles in the dispenser or sample tube.
4. open the bottom exit of the chromatographic column, start the pump, so that it is carried out at the set flow rate. The buffer should be allowed to flow slowly through the chromatographic column initially and then slowly increased to the final flow rate to avoid hydraulic impact on the formed column bed and avoid the column
The bed is not evenly formed. If the recommended pressure or flow rate is not reached, you can use the maximum flow rate of the pump you are using, which can also get a good filling effect. (Note: In subsequent chromatographic procedures, do not exceed *5% of the maximum column flow rate.) When the column bed height is stabilized, add at least 3 times the column bed volume of deionized water at the final column flow rate. Mark the height of the post bed.
5. Turn off the pump and close the outlet of the chromatographic column.
6. If a reservoir is used, remove the reservoir and place the distributor in the chromatographic column.
7. push the distributor to the column to the marked column bed height. Allow the loading fluid to enter the distributor and lock the distributor connector.
8. Connect the loaded chromatographic column to the pump or chromatographic system and start balancing. Re-adjust the distributor if necessary.

1.4 Sample purification
After Ni NTA Beads 6FF is filled, various conventional medium pressure chromatography systems can be used. The AKTA instrument is used as an example to introduce its use.
1. Fill the pump pipe with deionized water. Remove the upper plug, connect the chromatographic column to the chromatographic system, open the lower outlet, connect the preassembled column to the chromatographic system, and tighten.
2. Rinse the storage buffer with **5 times the column volume of deionized water.
3. Balance the column by Lysis Buffer at least 5 times the size of the column bed.
4. using pump or syringe sample. Note: The increased viscosity of the sample results in a large backpressure of the chromatographic column, even if the sample volume is small. Do not exceed the binding capacity of the column. The large sample volume can also cause a lot of back pressure, making the injector more difficult to use.
5. Wash the column with Wash Buffer until UV absorption reaches a stable baseline (usually at least ****5 column volumes). Note: Adding imidazole to the sample and binding buffer can increase the purity of the sample.
6. One-step or linear gradient Elution with Elution Buffer. In one-step elution, 5 times the column volume is usually sufficient. A small gradient, such as *0 times column volume or more, can be used to separate proteins with different binding strengths.

1.5 SDS-PAGE detection
Samples obtained from purified products (including effluent, wash and elution components) as well as original samples will be tested for purification using SDS-PAGE.

2. In-place cleaning
When the back pressure is found to be too high during the use of the filler or there is obvious contamination on the filler, it is necessary to carry out in-Place Cleaning operation (CIP). Before cleaning, remove Ni2+ (see 3. Regeneration of filler). After cleaning, store the filler in *0% ethanol, which is then rehung with nickel and stored in *0% ethanol. It is recommended to follow the steps below to remove residual contaminants from the filler, such as precipitated proteins, hydrophobic proteins, and lipoproteins.
Remove strongly hydrophobic binding proteins, lipoproteins and lipids
Such contaminants can be removed by cleaning ***0 column volumes with *0% isopropyl alcohol for a contact time of ****0 minutes. Then, it is cleaned with *0 times the column volume of deionized water. You can also choose to use an acidic or alkaline solution containing a stain remover, and the cleaning filler is 2 times the column volume. For example, a 0.1M acetic acid solution containing 0.**0.5% non-ionic detergent has a contact time of **2 hours. After the stain remover treatment, 5 column volumes need to be cleaned with *0% ethanol to completely remove the stain remover. Finally, use *0 times the column volume of deionized water to clean.
Remove proteins bound by ionic action
Use 1.5M NaCl solution for ****5 minutes for cleaning. Then *0 column volumes were cleaned with deionized water.

3. filler regeneration
The nickel ions contained in the histidine label protein affinity purification packings do not require frequent chelation to remove and reattach the nickel ions. When the back pressure is found to be too high during the use of the filler, there is obvious contamination on the filler, or the filler load is significantly lower, it is necessary to strip and re-hang nickel ions on the filler, that is, the filler regeneration. Load the filler into the appropriate chromatographic column and follow the procedure below to strip and rehang the nickel ions.
3.1 Use 0.2 M acetic acid solution (containing 6 M GuHCl) to clean 2 times the column volume;
3.2 Use deionized water to clean 5 times the column volume;
3.3 Use 2% SDS to clean 3 times the column volume;
3.4 Use deionized water to clean 5 times the column volume;
3.5 Use ethanol to clean 5 times the column volume;
3.6 Use deionized water to clean 5 times the column volume;
3.7 Use **0 mM EDTA (pH 8.0) to clean 5 times the column volume;
3.8 Use deionized water to clean 5 times the column volume;
3.9 Use **0 mM NiSO4 to clean 5 times the column volume;
3.*0 Use deionized water to clean *0 times the column volume;
After the filler is regenerated, it can be used immediately or stored in *0% ethanol at 4°C.

国家:	China
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位置:	China
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產品組 :	Protein Biology