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Purification & Detection

HIS-Select Convenient Formats


HIS-Select® iLAP® 5 ml Column, (H9913)

The HIS-Select iLAP (Integrated Lysis and Affinity Purification) 5 ml Column is a single-use, disposable column designed for one-step purification of histidine-tagged proteins directly from a 5 ml bacterial culture. This patent-pending technology allows simultaneous cell lysis and histidine-tagged protein capture in one easy step without the need for centrifugation of the bacterial culture. Each column contains 5 cell lysis/protein extraction reagent tablets as well as one HIS-Select Affinity Gel pellet. The lysis/extraction reagent tablets (CelLytic™ Express Tablets, C5491) include all the necessary detergents and enzymes needed for efficient cell lysis and protein extraction. The HIS-Select resin is highly selective for histidine-tagged proteins and reduces nonspecific binding. Each ready-to-use column is capable of purifying at least 1 mg of histidine-tagged protein in less than an hour. These columns are ideal for rapid colony screening for protein expression levels as well as for purifying functional protein for further downstream analysis. The resulting purified protein is compatible with functional protein assays, SDS-PAGE, Western blotting, and MALDI analysis.

Features & Benefits

  • Simultaneous one-step lysis and purification
  • Save time by eliminating cell harvest and lysate clarification steps
  • Preserve biological activity and achieve increased productivity
  • HIS-Select resin: Highly selective for histidine-tagged proteins
  • Ideal for rapid culture screening and determining protein expression levels

 

Efficient and Selective: High Purity and High Yield
GST-MAT iLAP Purification
HIS-Select<SUP>®</SUP> iLAP™ 5 ml Column
Lane
Sample Added
µl
1
Wide Range MW Marker (M4038) 3.5
2
BL21 E. coli TB culture expressing GST-MAT 10
3
iLAP Column Flow Through 10
4
iLAP Column Wash 10
5
iLAP Column Elution 10
Total protein in Elution = 2.52 mg

Figure 1: Recombinant GST-MAT (metal affinity tag) was expressed in BL21 E. coli cells. Five milliliters of bacterial culture were added directly to the HIS-Select iLAP column. The column was incubated, with mixing, for 15 minutes at room temperature. The resin was allowed to settle by gravity flow, and was washed twice with 2 ml of wash buffer (300 mM NaCl, 50 mM sodium phosphate, pH 8.0, 5 mM imidazole). Purified protein was eluted with 2 ml of elution buffer (300 mM NaCl, 50 mM sodium phosphate, pH 8.0, 200 mM imidazole). Total protein was quantitated using the Bradford assay.

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HIS-Select® High Flow Cartridges, (H7788), (H7163)

The HIS-Select High Flow Cartridges are pre-packed ready to use cartridges (column) that contain HIS-Select High Flow Nickel Affinity Gel (Product Number H0537). The cartridges are supplied in two sizes, 1.25 ml (Product Number H7788) and 6.4 ml (Product Number H7163). The cartridges’ luer lock fittings and accompanying adaptors make them suitable for Low Pressure Liquid Chromatography Systems (LPLC), Medium Pressure Liquid Chromatography Systems (MPLC), Fast Pressure Liquid Chromatography (FPLC™), and for use with a syringe. Plus, each pre-packed column binds >19 mg (1.25 ml column, Product Number H7788) or > 96 mg (6.4 ml column, Product Number H7163) of histidine-tagged recombinant protein.

Features & Benefits

  • Filled with Highly Selective HIS-Select High Flow Nickel Affinity Gel
  • Use at pressures up to 100 psi
  • Syringe or FPLC purification
  • Flow rates up to 5 ml/min

 

Poster: One Step Purification of HIS-tagged Fusion Proteins under High Pressure and High Flow Conditions (850 kb PDF)

HIS-Select<SUP>®</SUP> Cartridge

1     2     3      4     5     6     7     8      9     10    11    12   
Lane
Sample
1
3 µl Wide Range Marker (M4038)
2
Blank
3
3 µl Charge (GST-MAT)
4
Blank
5
3 µl Wash/Leak
6
Blank
7
3 µl Elution
8
5 µl Elution
9
Blank
10
5 µl Water Wash #1
11
5 µl Water Wash #2
12
Blank

Figure 1: Purification of recombinant protein using the HIS-Select™ High Flow Cartridge. Overnight cultures of transformed E. coli were induced with IPTG and recombinant GST-MAT was purified from a whole cell lysate (prepared with Cellytic B, Product Number B7435, containing Benzonase®, Product Number E1014) using the 6.4 mL high flow cartridge (Product Number H7163). The cartridge was washed using HIS-Select™ Wash Buffer (Product Number H5288) and bound proteins eluted using the HIS-Select™ Elution Buffer (Product Number H5413).

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HIS-Select® Cartridge, (H8286)

HIS-Select Cartridges are a conveniently pre-packed format that allows for rapid, small-scale purification of histidine-tagged proteins. Columns contain fittings that allow syringe purification, connection to FPLC or other medium pressure systems. Plus, each pre-packed column binds >15 mg of histidine-tagged recombinant protein.

Features & Benefits

  • Filled with Highly Selective HIS-Select Nickel Affinity Gel
  • Use at pressures up to 30 psi
  • Syringe or FPLC purification
  • Flow rates up to 5 ml/min

 

HIS-Select Cartridge

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HIS-Select® Spin Column, (H7787)

Pre-packed and ready-to-use HIS-Select Spin Columns allow for fast and consistent purification of small-scale crude cell extracts containing histidine-tagged proteins. The Spin Columns contain a matrix of silica particles and uses the HIS-Select patent-pending non-charged, hydrophilic nickel chelating linkage chemistry. HIS-Select Spin Columns are highly selective for histidine tagged proteins and exhibit very low non-specific binding of other proteins. These convenient Spin Columns allow for fast, one-step purification for the isolation of at least 150 µg of histidine-tagged protein in less than 15 minutes.

Features & Benefits

  • High Selectivity
  • Convenient and Ready-to-Use
  • Consistent protein binding each use
  • Compatible with CelLytic™ cell lysis reagents

 

Purification of Histidine-tagged protein from <i>E. coli</i> cell lysates using the HIS-Select™ Spin Column
Figure 1: Lysates from E. coli containing 300 µg Histidine-tagged protein were purified using a HIS-Select™ Spin Column and a competitor spin column.

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EZView™ Red HIS-Select® Nickel Affinity Gel, (E3528)

When performing small scale affinity capture, such as immunoprecipitation, the affinity matrix is difficult to see in the microcentrifuge tubes. Accidental aspiration of the resin leads to quantitative variability in results. The EZview Red Affinity Gels greatly reduce the risk of pellet loss. EZview resins perform as well as conventional non-colored affinity gel (Figure 1), but allow the user to easily differentiate pellet from supernatant. This correlates to more accurate data because less protein is lost.

 

  • Red color reduces the risk of incidental aspiration which improves recovery of target protein
  • Higher reproducibility due to more consistent yields

 

Figure 1:
Direct Affinity Capture of Tagged Proteins

Affinity capture of HAT-BAP fusion protein from
COS-7 cell lysates using HIS-Select and EZview
HIS-Select HC Nickel Affinity Gels
. Purified HAT-BAP fusion protein (lane 1) was spiked into COS-y lysates (107 cells in 1 ml RIPA buffer) at 5 µg/ml (lanes 4 and 5) and affinity captured with standard HIS-Select HC Nickel Affinity Gel (lane 5; Product Number E3528). Lanes 2 and 3 did not have HAT-BAP spiked in the lysates.

The blot shown was probed with anti-HAT antibody followed by goat anti-rabbit alkaline phosphatase conjugate secondary antibody (Product Number A3687) and visualized by BCIP/NBT substrate (Product Number B1911).

EZView tubes

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