Product FAQ

What is the cleavage recognition site for EKMax™ enterokinase? Do you offer a resin for removal of the enzyme after the cleavage?

Answer

EKMax™ enterokinase is a recombinant preparation of the catalytic subunit of bovine enterokinase. EKMax™ enterokinase recognizes the sequence DDDDK and cleaves the peptide bond after the lysine residue.

We offer EK-Away™ Resin that specifically binds EKMax™ Enterokinase, and can be used to remove it after cleavage.

Answer Id: E12958

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Product FAQ

How does SUMO protease work?

Answer

SUMO protease, also known as Ulp, is a recombinant fragment of ULP1 (Ubl-specific protease 1) from Saccharomyces cerevisiae. It is highly specific for the SUMO protein fusion, recognizing the tertiary structure of SUMO rather than an amino acid sequence. The SUMO protease itself has a His tag for easy removal from the protein mix after cleavage.

Answer Id: E12959

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Product FAQ

What enzyme should I choose if I do not want any extra amino acids left on my protein of interest after cleavage?

Answer

We would recommend using our SUMO system.

Answer Id: E12960

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Product FAQ

If my tissue has a high content of proteoglycans and/or polysaccharides, what can I do to ensure that these compounds don't contaminate the RNA I get from my TRIzol™ Reagent purification?

Answer

Pellet polysaccharides (also pellets genomic DNA): Centrifuge following homogenization before adding chloroform at 12,000 X g at 4 degrees C for 10 min to pellet polysaccharides. In addition, you may need to do a high-salt isopropanol precipitation as follows. After collecting the aqueous phase, add 0.25 mL isopropanol and 0.25 mL of 0.8 M sodium citrate, 1.2 M NaCl per 1 ml TRIzol™ Reagent. Mix the solution, centrifuge, and proceed with isolation as described. This precipitates the RNA and maintains proteoglycans and polysaccharides in a soluble form. Samples known to have a high content of proteoglycans or polysaccharides include rat liver, rat aorta, and plants.

Answer Id: E3189

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Product FAQ

Can I isolate my proteins based on their post-translational modification (PTM) or active binding site?

Answer

Enrichment of specific proteins or protein complexes can most easily be accomplished by using target-specific immobilized antibody or PTM specific affinity binding. We offer several kits targeting phosphorylated proteins, alpha- linked mannose and terminal glucose residues, N-acetyl glucosamine, polyubiquinated proteins, ATP binding sites, and active serine hydrolase enzymes. Choose the right protein enrichment kit for your application using the following link (http://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-purification/protein-enrichment.html).

Answer Id: E12961

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Product FAQ

My native eluted fractions do not contain the His-tagged protein when using the ProBond™ Putification System. Do you have any suggestions for me to try?

Answer

The lack of binding could be because the protein already eluted with the wash buffer. This means that the purification conditions were too stringent. To achieve less stringent conditions, try these options:

-Use only 10 mM or less imidazole (1-5 mM) in the binding or wash buffer and /or
-Reduce the NaCl concentration from 500 mM to 250 mM or less; a systematic titration may be necessary, i.e., reduce in increments of 100 mM and then fine tune.
-Try to use an imidazole step gradient.
-The His-tag is hidden due to folding; try a denaturing elution.

Answer Id: E12978

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Product FAQ

What is the sensitivity of the Imperial™ Protein Stain?

Answer

With the enhanced protocol, the Imperial™ Protein Stain can detect less than 3 ng protein per band in 3 hours.

Answer Id: E11074

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Product FAQ

What can I do to increase the RNA yield using TRIzol™ Reagent from small amounts of starting samples?

Answer

Add 10 micrograms of RNase-free glycogen to less than 10 mg tissue or less than 1 X 10e6 suspension cells. Glycogen, unlike salmon sperm DNA carrier, can be added when TRIzol™ Reagent is added to sample.

Answer Id: E3190

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Product FAQ

How does the Pierce™ Phosphoprotein Enrichment kit work?

Answer

The columns in the kit contain a proprietary metal that interacts with negative charges from phosphate groups. The optimized buffer conditions enable specific capture of phosphoproteins from complex biological samples.

Answer Id: E12962

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Product FAQ

My denatured eluted fraction does not contain the His-tagged protein when using the ProBond™ Purification System. Do you have any suggestions for me to try?

Answer

The protein may have been eluted because the procedure was too stringent. Reduce the stringency as follows:

-Increase the pH by 0.5 to 1.0 pH unit
-Decrease the NaCl concentration in increments of 50 to 100 mM

Answer Id: E12979

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Product FAQ

Is the Imperial™ Protein Stain compatible with mass spectrometry analysis?

Answer

Yes, here (https://tools.thermofisher.com/content/sfs/brochures/TR0050-Stained-gels-for-MS.pdf) is the procedure for processing gels stained with Imperial ™Stain, for mass spectrometry analysis.

Answer Id: E11075

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Product FAQ

How can I avoid genomic DNA contamination when isolating RNA with TRIzol™ Reagent?

Answer

If you will not need to isolate genomic DNA from the same sample and want to reduce the chance of gDNA contamination in your RNA, you should perform the optional centrifuge step mentioned in step 1 of the TRIzol™ Reagent manual prior to addition of chloroform.

After homogenizing your sample thoroughly in TRIzol™ Reagent, centrifuge the sample at 12,000 X g for 10 minutes at 4 degrees C. Genomic DNA, cellular membranes, and polysaccharides will form a pellet, and your RNA will be in the supernatant. Any lipids and fats in your sample may form a layer at the top of the solution as well. Remove the fat layer if necessary with a sterile tool and transfer the RNA supernatant to a new vial. Discard the DNA pellet.

Add chloroform to the RNA supernatant and proceed with the RNA isolation protocol.

To reduce gDNA contamination even more, you can treat your RNA after isolation with amplification grade DNase I. (Using non-amplification grade DNase I is not recommended, as it is not validated for absence of RNases and has been shown to degrade RNA samples in some cases.)

Answer Id: E3191

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Product FAQ

What is the expected phosphoprotein yield when using the Pierce™ Phosphoprotein Enrichment kit?

Answer

From 2 mg of total protein, the expected yield is 300 μg when using the Pierce™ Phosphoprotein Enrichment kit.

Answer Id: E12963

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Product FAQ

I'm seeing protein precipitation during native binding when using the ProBond™ Purification System. What do you suggest I do to avoid this?

Answer

Please see our suggestions below:

-Add 0.1% Triton™ X-100 or Tween®-20 to help solubilize further; purify at room temperature if protein is not temperature sensitive. However, keep in mind that most proteins are temperature sensitive.
-If secreted proteins are in media with low pH, they must be dialyzed to avoid Ni2+ reduction.
-If solubility is a real problem (e.g., microsomes), include up to 0.2% Sarkosyl in the 6 M guanidine lysis buffer; this will help to solubilize everything and may be still compatible with the ProBond™ or Ni-NTA purification column.

Answer Id: E12980

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Product FAQ

How do I prepare a solution of Coomassie R-250 dye?

Answer

Add 100 mL of glacial acetic acid to 450 mL.
Dissolve 3 g of Coomassie R-250 dye in 450 mL methanol.
Mix the acetic acid and methanol solutions.
Filter the solution before use.

Answer Id: E13303

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