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Can I use protease inhibitors for mass spectrometry (MS) sample prep? Product FAQ

Answer

We don't recommend adding protease inhibitors as they can affect trypsin and trypsin/Lys-C activity. If protease inhibitors are present in the protein sample or cells, they should be removed by dialysis, diafiltration, desalting, or protein precipitation prior to enzymatic digestion. For phosphopeptide enrichment workflows, we recommend adding phosphatase inhibitors to the lysis solution prior to cell lysis.

Answer Id:: E18854

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What crosslinker can be used to link a compound through a phenolic hydroxyl group (or tyrosyl group) to a protein? Product FAQ

Answer

Our PMPI crosslinker (Cat No. 28100) is a maleimide-and-isocyanate crosslinker for attaching compounds to sulfhydryl groups after conjugating the linker to hydroxyl groups by a urethane (carbamate) bond. The first reaction is conjugating the hydroxyl group with the isocyanate. This will create a maleimide-activated molecule. 

You could then attach thiol groups on the protein using 2-Iminothiolane (Product No. 26101) or SATA (Product No. 26102). Remove excess reagent by dialysis or desalting and then react with the maleimide-activated molecule in the first step above.

Answer Id:: E15682

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What are the general characteristics of pyridyl disulfides? Product FAQ

Answer

Pyridyl disulfides react with sulfhydryl groups over a broad pH range to form disulfide bonds. As such, conjugates prepared using these crosslinkers are cleavable with typical disulfide reducing agents, such as dithiothreitol (DTT).

During the reaction, a disulfide exchange occurs between the –SH group of the target molecule and the 2-pyridyldithiol group of the crosslinker. Pyridine-2-thione (MW 111; λmax 343nm) is released as a byproduct that can be monitored spectrophotometrically and removed from protein conjugates by dialysis or desalting.

Answer Id:: E15715

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What can be done if many protein conjugates are formed when I add crosslinkers to cell suspensions or cell lysates? Product FAQ

Answer

- Choose more selective heterobifunctional crosslinkers with photo-reactive groups, which can be made to react at selected times and only in response to irradiation by UV light. Such crosslinkers with a thermoreactive group (spontaneously reactive) at one end and a photo-reactive group on the other end can be reacted first with a protein through the thermoreactive end. The labeled protein that can be used as bait for other interacting proteins. The modified protein is introduced into a sample and allowed to interact with other proteins. Then, the sample is exposed to UV light, which causes the photo-reactive end of the modified protein to covalently link to nearby molecules, thus “freezing” in place any interacting protein as a complex.
- Remove non-reacted reagent by dialysis or desalting.

Answer Id:: E15636

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What are the general characteristics of N-hydroxysuccinimide esters (NHS Esters)? Product FAQ

Answer

NHS esters are reactive groups formed by EDC-activation of carboxylate molecules. NHS ester-activated crosslinkers and labeling compounds react with primary amines in slightly alkaline conditions (pH 7.2-8.5) to yield stable amide bonds. The reaction releases N-hydroxysuccinimide (MW 115), which can be removed easily by dialysis or desalting. Primary amine buffers such as Tris or TBS are not compatible because they compete for reaction; however, in some procedures, it is useful to add Tris or glycine buffer at the end of a conjugation procedure to quench (stop) the reaction.

Sulfo-NHS esters are identical to NHS esters except that they contain a sulfonate (–SO3) group on the N-hydroxysuccinimide ring. This charged group has no effect on the reaction chemistry, but it does tend to increase the water-solubility of crosslinkers containing them. In addition, the charged group prevents sulfo-NHS crosslinkers from permeating cell membranes, enabling them to be used for cell surface crosslinking methods.

Answer Id:: E15711

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What is the difference between your dialysis devices (plates, device, cassettes, flasks)? Product FAQ

Answer

Please view our selection table to choose the right dialysis device for your experiment - https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-dialysis-desalting-concentration/dialysis-products.html.

Answer Id:: E12818

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How do I select the right Pierce Protein Concentrator for my experiments? Product FAQ

Answer

Please view our selection table (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-dialysis-desalting-concentration/protein-concentrators.html) to choose the right protein concentrator to fit your needs.

Answer Id:: E12848

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I am planning to perform in vivo siRNA delivery. I would like to dialyze my complexes, what do you recommend I use? Are there other options for preparing complexes for in vivo transfection? Product FAQ

Answer

Complexes prepared with Invivofectamine 3.0 Transfection Reagent do not require either diafiltration or dialysis. But if dialysis is needed, any of the Slide-A-Lyzer dialysis systems may be used, as long as the MW cutoff is 50 kDa. At the end of dialysis, you may observe some volume increase. If volume reduction is needed, follow dialysis with a Pierce Protein Concentrator but ensure the final volume is no less than the total undiluted complex volume to avoid binding issues to the membrane. Alternatively, dilute the complex with PBS by 6-fold and directly concentrate until the total volume is back to no less than the starting volume.

Please go to our our dialysis, desalting, and concentration selection guide here: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-dialysis-desalting-concentration.html

Answer Id:: E9056

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I want to use an Alexa Fluor Antibody Labeling kit to label my antibody. The antibody has histidine and glycine in the stock solution. Will this be a problem? Product FAQ

Answer

Yes. The reactive dye in the kit reacts with primary amine groups on histidine and glycine. This can result in less labeling of the antibody. The histidine and glycine must be removed, such as with dialysis or a desalting column. Other components that can interfere include BSA, gelatin, and Tris buffers.

Answer Id:: E6419

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What is the suggested method for concentrating protein following purification from ProBond resin? Is dialysis necessary to get rid of the imidazole? Product FAQ

Answer

Concentration and dilution through a Pierce Protein Concentrators protein concentration filter (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-dialysis-desalting-concentration/protein-concentrators.html) will both concentrate the protein and remove the imidazole all in one step. Dialysis is not necessary.

Answer Id:: E3665

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What sizes of Pierce protein concentrators do you offer? Product FAQ

Answer

The products are available in 4 sizes (0.5 mL, 6 mL, 20 mL, and 100 mL) with a range of molecular weight cutoffs (3K, 5K, 10K, 30K, and 100K). See our selection chart - https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-dialysis-desalting-concentration/protein-concentrators.html.

Answer Id:: E12870

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I am planning to perform in vivo siRNA delivery. If I plan to diafiltrate the complexes, what do you recommend I use? Product FAQ

Answer

Complexes prepared with Invivofectamine 3.0 do not require either diafiltration or dialysis. However, if diafiltration is used, any of the Pierce Protein Concentrator PES systems may be used, as long as the MW cutoff is 50 kDa. Keep the final volume to no less than the total undiluted complex volume. Please go to our concentrator selection guide here: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-dialysis-desalting-concentration/protein-concentrators.html

Answer Id:: E9057

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What are the different sizes of PES protein concentrators you offer? Product FAQ

Answer

Pierce polyethersulfone (PES) protein concentrators are offered with six distinct molecular weight cutoffs (MWCOs) of 3K, 5k, 10K, 30K, 50K, and 100K meant for processing four different sample volumes ranges viz., 100-500 µL, 2-6 mL, 5-20 mL, and 20-100 mL, respectively. Please see our selection chart and video at: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-dialysis-desalting-concentration/protein-concentrators.html

Answer Id:: E17294

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What should I do to increase the coupling efficiency of antibodies to the Pierce NHS-Activated Magnetic Beads? Product FAQ

Answer

-Make sure all primary amine-containing buffer components of the buffer like Tris or glycine are completely removed by dialysis, desalting, or the use of the Pierce Antibody Clean-Up Kit (Cat. No. 44600) before coupling to magnetic beads
-Make sure the antibody is mixed with the NHS-beads IMMEDIATELY after washing the beads. A delay may cause hydrolysis of the NHS groups on the resin which leads to low coupling efficiency.
-Finally make sure the pH of the non-amine-containing coupling buffer used for coupling is between pH 7-9

Answer Id:: E13055

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Is the final soluble nuclear fraction dialyzable when using the NE-PER Extraction reagents? Product FAQ

Answer

Yes. The nuclear fraction can be dialyzed in a Slide-A-Lyzer MINI Dialysis Unit (Cat. No. 69550) against a buffer, such as PBS, that is compatible with the specific downstream application. A buffer exchange can also be performed using the Thermo Scientific Protein Desalting Spin Columns (Cat. No. 89849) or Zeba Desalt Spin Columns (Cat. No. 89882).

Answer Id:: E8196

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