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General FAQs
Does the ProMTag permanently modify tagged proteins?
No! The ProMTag linkage is completely reversible and proteins are left in their original, unmodified state following ProMTag reversal.
What are RC tubes and how do I use them?
Resin Capture (RC) tubes are designed with a small slit in the bottom that allows for the passing of liquid with very little dead volume, but retains the solid resin. The RC tubes have two main features: a slit at the bottom of the tube, and a vent hole at the top. These tubes function similarly to a typical spin column, but require only brief (<5 seconds) centrifugation steps to fully spin out the liquid. When using the RC tube, take care to avoid touching the slit, and always spin and vortex the RC tube in an adapter tube.
What is special about MT-Trypsin?
MT-Trypsin is a methyltetrazine (MT) labeled trypsin that is stabilized, resistant to autolysis, and capturable by TCO resin. This allows for short digestion times (1 hour) at high MT-Trypsin concentration without the risk of overwhelming the final sample with trypsin autolysis peptides. During a 1 hour digestion period, MT-Trypsin will completely digest input proteins and also be captured on the TCO resin.
ProMTag IP-to-MS FAQs
Does the ProMTag permanently modify tagged proteins?
No! The ProMTag linkage is completely reversible and proteins are left in their original, unmodified state following ProMTag reversal.
Can I use my lysis buffer with the ProMTag IP-to-MS method?
While we recommend utilizing our IP lysis buffer, other lysis buffers are compatible with the method. You can use your own lysis buffer as long as it does not contain TRIS (or any other buffer with primary amines) and is ~pH 8.0. If your lysis technique uses TRIS, we recommend switching to 100 mM HEPES pH 8.0. Note that the IP-to-MS IP-LB is a gentle lysis buffer with added protease inhibitors. If you use your own lysis buffer, ensure it is compatible with immunoprecipitation.What protease inhibitors can I use in my lysis buffer to prevent protein degradation?
We recommend supplementing your lysis buffer with phenylmethylsulfonyl fluoride (PMSF), pepstatin, and leupeptin. If another set of protease inhibitors is used, it is important to ensure those inhibitors do not contain primary amines, which will interfere with ProMTag labeling.Can I mechanically grind or sonicate my sample to improve lysis?
Yes, you can use sonication and any mechanical homogenization method available to improve the lysis of viscous lysates and dense tissues. The IP lysis buffer does not contain harsh denaturants, so it is very important to ensure proper sample lysis. For sonication, we recommend sonicating for 30-40 blasts at 30% power and 30% duty cycle while keeping the sample on ice.How much protein lysate is required for the ProMTag IP-to-MS workflow?
You need 100 µg of protein lysate per sample and the lysate concentration should not be lower than 0.5 mg/mL.What antibody sources are compatible with the ProMTag IP-to-MS method?
We have tested the ProMTag IP-to-MS method with serum, plasma, bronchoalveolar lavage fluid (BALF), and purified antibodies.What is the required amount of serum or antibody source for the ProMTag IP-to-MS method?
The ProMTag IP-to-MS workflow requires 2-5 µg of purified antibody of your choice, or 10 µl of plasma or serum, or 50 µl of bronchoalveolar lavage fluid (BALF).How long does it take to complete the ProMTag IP-to-MS workflow?
The entire workflow takes 6-8 hours to complete depending on the number of samples being processed. About an hour and a half of hands-on time is required.ProMTag Multiomics FAQs
Does the ProMTag permanently modify tagged proteins?
No! The ProMTag linkage is completely reversible and proteins are left in their original, unmodified state following ProMTag reversal.
What additional reagents and equipment are needed before I start using the ProMTag Multiomics kit?
The required reagents not provided in the ProMTag Multiomics kit are: 2-Mercaptoethanol, DNase and RNase enzymes if DNase and/or RNase treatment is necessary for intended downstream applications, RNase inhibitor (optional, recommended for RNase-rich tissues), and ultrapure, nuclease-free deionized water. You also need to provide your starting sample (tissue, cells, lysate, etc.). The workflow requires basic laboratory equipment including a benchtop centrifuge (mini or full size), a sample rotator (rotisserie or carousel), a heating block, and a vortex.Can I use my lysis buffer with the ProMTag Multiomics kit?
While we recommend utilizing the lysis buffer included with the ProMTag Multiomics kit, other lysis buffers are compatible with the kit. You can use your own lysis buffer as long as it does not contain TRIS (or any other buffer with primary amines) and is ~pH 8.0. If your lysis technique uses TRIS, we recommend switching to 100 mM HEPES pH 8.0. If your lysis buffer does not contain a strong denaturant, we highly recommend using a protease inhibitor in your lysis buffer to prevent protein degradation. We also highly recommend utilizing a lysis buffer containing a high concentration of guanidine thiocyanate to help maintain RNA integrity. The provided lysis buffer must be supplemented with 2-Mercaptoethanol (not included) to a final concentration of 284 mM immediately prior to use.Can I mechanically grind or sonicate the tissue to improve lysis?
You can use any mechanical homogenization method. However, you should avoid sonication as it will result in degradation of nucleic acids.Which protein quantification methods are compatible with the lysis buffer provided with ProMTag Mutiomics kits?
A Bradford protein assay can be used to calculate the protein content of the lysate. This lysis buffer contains 2-mercaptoethanol and is incompatible with BCA assays.Can I use a higher sample volume with the ProMTag Multiomics method if my sample is dilute?
We recommend keeping the input volume at or under 50 µL. If your sample is dilute, a starting volume of up to 100 µL can be used. However, RNA and DNA recovery may be negatively affected by high input volumes.How long does it take to complete the ProMTag Multiomics workflow?
The entire workflow takes 5-6 hours to complete depending on the number of samples being processed. About an hour and a half of hands-on time is required.Is the ProMTag Multiomics method compatible with cells and tissues stored in RNAlater® solution?
Yes, the ProMTag Multiomics method is compatible with samples stored in RNAlater®. However, yields with these samples may be slightly lower than with fresh samples.What are the expected peptide and nucleic acid yields with the ProMTag Multiomics kit?
If you start with 100 µg of protein from a cultured cell lysate, the average yields are: 0.35 µg DNA, 2.40 µg RNA, and 10.42 µg peptide. For more details and the yield information for mouse tissues please refer to our poster (Biedka et al., HUPO 2023) and Supplementary Table 2 in our paper (Biedka et al., Commun. Biol. 2024) available in the Resources section of the website menu.What peptide and nucleic acid quantification methods are compatible with the ProMTag Multiomics kit?
You can use any available peptide or nucleic acid quantification methods. We recommend using Pierce™ Quantitative Fluorometric Peptide Assay to measure peptide amount Qubit™ RNA broad-range assay kit for RNA and Qubit™ dsDNA broad-range assay kit for DNA quantifications.Is the ProMTag Multiomics kit suitable for RNase-rich tissues?
Yes, you can use our Mutiomics kit to isolate RNA from cells and tissues that have high RNase activity. The lysis buffer provided with the kit was formulated to help maintain RNA integrity. If your sample is RNase-rich, we recommend supplementing NAE1 with RNase inhibitor immediately before the first nucleic acid elution step.ProMTag Proteomics FAQs
Does the ProMTag permanently modify tagged proteins?
No! The ProMTag linkage is completely reversible and proteins are left in their original, unmodified state following ProMTag reversal.