Product FAQ

What caspase does the ApoTarget™ Caspase Colorimetric Protease Assay kit recognize?

Answer

The kit provides a simple and convenient means for quantitating the enzyme activity of caspases that recognize the amino acid sequences VDVAD (caspase-2), DEVD (caspase-3), VEID (caspase-6), IETD (caspase-8), and LEHD (caspase-9).

Answer Id: E10093

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Product FAQ

What is the difference between NucBlue™ Live reagents stain and NucGreen™ Dead reagents stain?

Answer

The NucBlue™ Live reagent stains the nucleus of all cells, while NucGreen™ Dead reagent stains only the nuclei of dead cells.

Answer Id: E10094

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Product FAQ

What are the advantages of using the Click-iT™ Plus EdU Imaging kits compared to traditional BrdU assays?

Answer

The Click-iT™ Plus EdU kits use a small size of picolyl azide dye that allows for efficient detection of the incorporated EdU using mild conditions. Standard aldehde-based fixation and detergen permeabilization are sufficient for the reagent to gain access to the DNA. Additionally, EdU Assay kits are easy to use and fully compatible with DNA staining, including dyes for cell cylce analysis. The kit can also be multiplexed with fluorescent proteins.

Answer Id: E10095

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Product FAQ

Can I purify proteins from my mammalian tissue culture media directly on the ProBond™ resin?

Answer

This can be achieved, though not effectively. DMEM and other mammalian tissue culture media contain many contaminating proteins (and free amino acids like glutamine and histidine) that may compete for binding to the ProBond™ resin. For best results, mammalian tissue culture media should be dialyzed (against binding buffer) or filtered prior to loading on equilibrated ProBond™ resin. Other methods such as ion exchange or sizing columns can help to prepare the protein for effective isolation on the ProBond™ column. If necessary, the culture media may be adjusted to facilitate direct binding to the column. The pH of the media should be adjusted to 7.5-8.0. The salt concentration in the media should be 0.5 M NaCl.

Answer Id: E12950

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Product FAQ

What can the BioParticles™ product line be used for?

Answer

These fluorescent Bioparticles products have been employed to study phagocytosis by fluorescence microscopy, quantitative spectrofluorometry, and flow cytometry. We offer E.Coli, S. aureus, and zymosan BioParticles conjugates covalently labeled with a variety of different fluorophores.

Answer Id: E10096

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Product FAQ

How does the Amplex Red Neuraminidase (Sialidase) Assay kit detect neuramimidase activity?

Answer

The assay utilizes Amplex Red to detect H202 generated by galactose oxidase oxidation of desialiated galactose, the end result of neuraminidase action. The H202, then, in the presence of HRP, reacts with a 1:1 stiochiometry with Amplex Red reagent to generate the red-fluorescent oxidation product, resorufin.

Answer Id: E10113

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Product FAQ

Do you have a protocol for purification of His-tagged proteins synthesized in the ExpressWay™ Cell-Free Expression System?

Answer

Purification may be performed at 4 degrees C or room-temperature depending upon the sensitivity of the synthesized product.

1. Upon completion of incubation, remove the desired portion of reaction for His-tag purification to a clean microcentrifuge tube. Add 4 volumes of Binding buffer and vortex briefly (Add 200 μL for 50 μL of reaction). Centrifuge 5 minutes at 12,000 rpm.
2. Transfer the supernatant to a 2.0 mL tube containing 50 μL pre-equilibrated resin.
3. Incubate with shaking or mixing for 30-60 minutes.
4. Spin down resin for 2 minutes at 800 x g. Do not spin any higher or the resin will collapse and recovery will be low. Carefully remove supernatant.
5. Add 200 μL wash buffer and mix for 5 minutes.
6. Spin down resin for 2 minutes at 800 x g. Carefully remove supernatant.
7. Repeat steps 5 and 6.
8. Add 100 μL Elution Buffer and mix for 5 minutes.
9. Spin down resin for 2 minutes at 800 x g. Carefully remove and save supernatant.
10. Repeat steps 8 and 9.

Binding Buffer:
50 mM NaP04, pH 7.0
500 mM NaCl
6 M guanidine HCl (optional)**

Wash Buffer:
50 mM NaP04, pH 7.0
500 mM NaCl
15-25 mM imidazole*

Elution Buffer:
50 mM NaP04, pH 7.0
500 mM NaCl
150-250 mM imidazole*

**Depending on downstream applications, the purification may be performed under semi-denaturing conditions, or native conditions. Under semi-denaturing conditions, dilute the reaction in denaturing Binding Buffer containing 6 M guanidine HCl; then wash and elute with native buffers.

The concentration of imidazole is dependent upon the type of resin used. For Ni-NTA or ProBond™ resins, use 25 mM imidazole in the wash buffer and 250 mM imidazole in the elution buffer.

Answer Id: E12951

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Product FAQ

What optical filter do your recommend with the BacLight™ Green Bacterial Stain?

Answer

We recommend filters suitable for fluorescein with the BacLight Green kit.

Answer Id: E10097

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Product FAQ

How long does the labeling take when using the RadPrime DNA Labeling system?

Answer

Typically labeling is complete in 10 minutes, and purifiation of the probe is not usually required.

Answer Id: E10114

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Product FAQ

Do you have a protocol for purification of His-tagged proteins from E. coli lysate?

Answer

When purifying His-tagged proteins proteins from E. coli lysates, keep in mind that there is a 29 kDa endogenous protein SlyD. SlyD has a histidine-rich c-terminus and is found in all strains of E. coli and Salmonella. The contamination is apparent when the His-tagged protein is expressed at a low level or not expressed at all. In such cases, SlyD will bind to the nickel column with great affinity. Increase the purification stringency to overcome SlyD binding.
If protein is released into LB media from E. coli, try native isolation conditions. Dialyze against binding buffer and possibly concentrate before going on to the ProBond™ resin (10% glycerol in the dialysis binding buffer will concentrate the secreted protein well). Another option is to add about 24 g NaCl and 2.8 g Na2HPO4 per liter of media, and adjust the pH to 7.8 with NaOH or HCl. This will turn the media into pseudo-binding buffer (~500 mM NaCl, ~20 mM NaPO4, pH 7.8); perform binding, washing, and eluting with either imidazole or by altering pH.

Answer Id: E12952

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Product FAQ

What is the reaction product excitation/emission maxima when using the EnzChek™ Phosphatase Assay kit?

Answer

The reaction product has excitation/emission maxima of approximately 358/455 nm. Fluorescence can be measured in a fluorescence microplate reader or a standard fluorometer.

Answer Id: E10098

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Product FAQ

What is the approximate excitation/emission maxima of the reaction product when using the EnzChek™ Direct Phospholipase C Assay kit?

Answer

The approximate fluorescence excitation/emission maxima is: 509/516 in nm.

Answer Id: E10115

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Product FAQ

What Ion PGM™ chips are available? How much sequencing data can I expect from each chip type?

Answer

The Ion PGM™ System supports the Ion 3 series chips, including the Ion Torrent™ Ion 314™ Chip Kit v2 (Cat. No. 4482261), Ion Torrent™ Ion 316™ Chip Kit v2 (Cat. Nos. 4483188 and 4483324), and Ion Torrent™ Ion 318™ Chip Kit v2 (Cat. Nos. 4484354 and 4484355). The Ion 314™ Chip Kit v2 is available in an 8-pack; the Ion 316™ Chip Kit v2 and Ion 318™ Chip Kit v2 are available in 4-packs and 8-packs.

For more details, please see the Ion PGM™ System Specification Sheet (https://tools.thermofisher.com/content/sfs/brochures/PGM-Specification-Sheet.pdf).

Answer Id: E10454

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Product FAQ

I'm seeing air bubbles in my affinity purification column. What's the best way to get rid of the air bubbles to restore flow rate?

Answer

Please see this Tech Tip (http://tools.thermofisher.com/content/sfs/brochures/TR0007-Remove-air-bubbles.pdf) for suggestions.

Answer Id: E12970

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Product FAQ

Do you have suggestions for purification of secreted His-tagged proteins from E. coli culture?

Answer

A simple and reliable method for precipitating protein from bacterial culture supernatants based on a pyrogallol red- molybdate-methanol (PRMM) protocol has been developed and applied for the analysis of proteins secreted by a bacterial type III secretion system (Caldwell RB, Lattemann CT (2004). Simple and reliable method to precipitate proteins from bacterial culture supernatant Appl Env Micro 70(1):610-612)(http://aem.asm.org/content/70/1/610.full.pdf).

Answer Id: E12953

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