Lifesensors FAQ

Please use the Contact Us form under the About Us tab, where you can provide your contact information and details about your project. 

Please include which target protein(s) you wish to study, the quantity of compounds you wish to test, the type of assay you wish to use (if known), and any other important details of your scope of work.  

Alternatively:  

Contact Customerservice@lifesensors.com for product and technical inquiries. 

Contact bd@lifesensors.com for CRO services, custom product/service requests, and other custom inquiries. 

Yes, LifeSensors has many international distributor partners. International customers (non-US) should contact their local distributor for product orders. For a list of LifeSensors distributors, visit our Distributor Page.  

Details on the protocol for the product or assay kit can be found on each product page under ‘Documents’. Additional information and technical help are available by contacting customerservice@lifesensors.com.  

Product datasheets can be found on each product page under the ‘Documents’ tab.  

TUBEs and recombinant proteins should be limited to minimal freeze-thaw cycles. Once received, the product should be aliquoted appropriately based on usage and stored in the appropriate temperature as stated on the website.  

To convert mg/mL to molar concentration, you will first need to acquire the molecular weight of the protein. You then divide the concentration by the molecular weight.  

For example, wild type ubiquitin has a molecular weight of 8.56 kDa. Molar concentration of a 5.0 mg/ml solution of wild type ubiquitin can be calculated as follows; 5.0/8.56= 0.584 mM or 584.0 µM. 

Recombinant proteins should be stored at -80°C. 

We can receive compounds in powder form, as DMSO stocks or as aqueous solutions. Compounds can be shipped to us as individual vials or in 96-well or 384-well plates.  

Sample shipment address: 

LifeSensors, Inc. 

ATTN: Karteek Kadimisetty 

271 Great Valley Parkway 

Malvern, PA 19355 

Yes. We can provide E3 or DUB with custom protein tag or custom labeling. Please contact bd@lifesensors.com for custom product/service requests.

Please contact bd@lifesensors.com for licensing inquiries.

LifeSensors’ TUBE (Tandem Ubiquitin Binding Entity) technologies are industry-leading tools to detect and enrich/purify poly-ubiquitylated proteins from cell lines, tissues, and organs. LifeSensors has two commercially available pan-TUBEs (TUBE1 and TUBE2). Both TUBE1 and TUBE2 come in various flavors such that they can be used for far-Western detection (FLAG, Biotin), enrichment/purification (linked to Agarose and Magnetic beads), and in vivo imaging (TAMRA, FITC-attached) applications. For more information, please see our TUBEs page.

TUBE1 and TUBE2 are made from different ubiquitin-binding domains (UBDs). However, both of them have been shown to bind all the linkages of polyUb with single digit nM Kd. TUBE1 has been shown to preferentially bind K63-polyUb over K48-polyUb. TUBE2 binds both K48- and K63-polyUb with equal affinities.

Yes, LifeSensors sells a number of linkage-selective TUBEs (so far M1, K48, and K63). Like pan-TUBEs, these linkage-selective TUBEs also come in various flavors such that they can be used in far-Western detection, enrichment/purification, and imaging applications.

LifeSensors have recently developed a new K48 TUBE HF (high-fidelity) that has comparable affinity (Kd of ~20 nM for tetra-ubiquitin) to their cognate poly-Ub as our existing K63 TUBE and M1 TUBE. The old K48TUBE binds tetra-ubiquitin with a Kd of ~200 nM.

TUBEs bind to ubiquitin/polyubiquitin chains which are highly conserved across all eukaryotes. However, the poly-ubiquitin linkage type (K48, K63) and the relative abundance of target protein should be considered while making a selection of TUBEs for your experiments.

No, TUBEs have not been tested by LifeSensors for organisms other than mammalian and yeast ubiquitin/poly-ubiquitins. Since ubiquitin is highly conserved from yeast to human, theoretically TUBEs should work for plants and other organisms. We welcome any feedback from customers who have studied other organisms with our TUBEs.

The abundance of the target protein and the type of sample (cells, tissues, organs) should be considered to estimate the amount of cell/tissue extracts needed. As a starting point, we recommend to use 20 µL of agarose -TUBE beads or 100 µL magnetic-TUBE slurry per milligram of cell extract. The amount of cell/tissue extracts, volume of the eluate, and the volume of the agarose/magnetic beads should be optimized by the end user.

Elute poly-ubiquitylated proteins from agarose/magnetic-TUBEs using our proprietary elution buffer (LifeSensors Cat # UM411B). Alternatively, for gel-based separation and subsequent applications, the ubiquitylated proteins can be eluted using a standard loading dye for SDS-PAGE.

Positive control: How do I know that TUBEs work?
The properties of TUBEs have been well characterized and published. Each TUBE lot is quality controlled for overall binding capacity and the pull-down efficiency of polyubiquitylated proteins using cell lysate.

Negative control: How do I know that TUBEs aren’t yielding an artifact?
We recommend to include a negative control in your pull-downs that contains the quenched agarose resin (LifeSensors cat # UM400).

The UbiTest platform replaces traditional immunoblotting methods for measuring cellular ubiquitylation, enabling simple comparisons of substrate ubiquitylation levels between samples and determination of ubiquitin chain linkage types. UbiTest is based on TUBE technology for enriching polyubiquitylated proteins. By visualizing the ubiquitylated fraction both with and without DUB digestion of polyubiquitin chains, UbiTest streamlines the confirmation of ubiquitylation status. For more information about UbiTest, please also visit our UbiTest product pages, where you can purchase off-the-shelf assay kits for performing UbiTest in your own lab.

LifeSensors has also developed High-Throughput (HT) UbiTest which is a customized medium-throughput assay for absolute quantification of ubiquitylated substrate levels in cells that combines UbiTest principles and TR-FRET technology. Please contact us if you like to develop similar assays for your target protein.

Yes, LifeSensors offers TUBE-based proteomics services. LifeSensors have developed two proprietary technologies to help researchers accomplish their scientific goal – (1) identify cellular proteins that are ubiquitinated and characterize the ubiquitin-linkages on them (TUBE proteomics), and (2) qualitative and quantitative profiling of ubiquitination sites on proteins (UbiSight proteomics). For more information, please contact us or visit our service pages.

Magnetic beads should be stored at 4°C after arrival. After use any unused beads should be stored with PBS + 20% EtOH.  

Agarose beads should be stored at 4°C after arrival. Do not freeze the agarose beads.

Unlike with whole cell lysate, probing for a housekeeping protein, like GAPDH, is not possible after TUBE enrichment. Thus, we recommend completing pulldowns from all experimental conditions using the same quantity of lysate and the same amount of TUBE. 

Using a matrix in TUBE pulldowns provides a solid support for specific interactions, enabling high specificity and efficient ubiquitinated protein enrichment. Solution pull-down methods offer flexibility in studying protein interactions in a more native environment. This approach allows for the investigation of dynamic interactions between proteins without the constraints of being bound to a solid support. It’s particularly useful when studying transient or weak interactions that might be disrupted in the rigid conditions of a solid-phase matrix. 

Magnetic TUBEs offer various advantages over agarose TUBEs. Agarose TUBEs require centrifugation steps for separation of beads from the solution phase. Magnetic beads offer an advantage in terms of ease of separation, as well as their ability to be used in bead-based ELISAs. 

High-Capacity TUBE1 (UM501M) were designed by coating polymeric high-capacity magnetic beads to allow superior enrichment of polyubiquitinated proteins along with minimizing non-specific binding to proteins in tissue and cellular lysates. Magnetic TUBE1 beads (UM401M) are 1 µm and High-Capacity TUBE1 beads (UM501M) are 2.8 µm in diameter. 

Depending on the type of TUBE purchased, the protocol differs slightly. Please refer to the manual for each specific product when performing a pulldown. This manual can be found on the product page for each specific TUBE. 

For western blotting, to detect overall ubiquitination signal, use VU-1 antibody. To detect target ubiquitination, use a target specific antibody.

To reduce background and non-specific binding, we recommend preincubating the TUBEs in 3% BSA for one hour at 4 °C followed by three washes with PBST. 

Yes. TUBEs can be used as detection reagents in both Western blotting and ELISA-based assays. LifeSensors offers an HRP-conjugated TUBE2 for easy, one-step detection of pan-ubiquitination. Additionally, we offer biotin-labeled TUBEs (M1 Biotin TUBE, TUBE1 Biotin, TUBE2 Biotin, K63 Biotin TUBE, and K48 HF Biotin TUBE) for detection with streptavidin-HRP. 

Many engineered/designer proteins or single-chain antibodies are poorly expressed. Fusion with SUMO/SUMOstar enhances the stability, solubility, and yield of a protein. SUMO is one of the most conserved proteins in eukaryotes and have been shown to post-translationally modify a number of proteins in cells. SUMOylation plays an important role in various cellular process such as nuclear-cytosolic transport, transcriptional regulation, and protein stability. SUMO/SUMOstar-fusion leads to a dramatic enhancement of expression in E. coli, yeast, insect, and mammalian cells and promotes correct folding and hence biological activity. Additionally, this system simplifies and lowers the costs associated with protein expression and purification. Using this technology, a plethora of proteins have been shown to enhance their expression, stability, and yield.

SUMO has been used with a range of proteins for a variety of purposes. We included a table attached to our SUMO technology page that has a range of proteins, as well as additional information from each scientific publication. SUMO system has been used at more than a thousand labs around the world. Almost all members of each protein family have been expressed using the SUMO system in a variety of hosts. We encourage reaching out, as we have included a partial list of references, but our library is growing constantly in this cutting edge of research.

We offer systems that are optimized for:
– Bacterial: E.coli
– Yeast: S.cereviseaand P.pichia
– Baculoviral/Insect cell system
– Mammalian: HEK293and CHO cells

Attachment of the C-terminus of SUMO (Small Ubiquitin Like Modifier) to the N-terminus of a protein of interest can dramatically improve protein solubility, achieve native protein folding, and increase total yield by improving expression and decreasing degradation. LifeSensors protein expression system has pioneered SUMO fusion tag technology, which increases the quantity and quality of numerous recombinant proteins in both prokaryotic and eukaryotic expression hosts.  Our cleavable SUMO tag system allows production of recombinant proteins with their native N-terminus intact and without any unwanted residues. We offer a protein expression service related to the expression and purification of proteins.

Please contact bd@lifesensors.com for licensing inquiries.

For cell lysis, use RIPA buffer with 1 mM PMSF, 20 µM MG132, 50 µM PR619, 5 mM 1,10 phenanthroline, Protease inhibitor cocktail 1 to 500 dilution (Sigma Cat#P8849).

No! LifeSensors’ plates work for bivalent and trivalent PROTACs as well as molecular glues.

The E3 ligases offered for PA770 In vitro ubiquitination assay are Cereblon, VHL, and HDM2. Custom Kits can be created for other E3 Ligases as a separate offering.

We offer mouse and rabbit secondary antibodies. Which one you use will depend on your primary antibody and in which organism it was raised.

DUBTACs, or DeUBiquitinating TArgeting Chimeras, recruit DUBs to a target protein and remove ubiquitin chains, resulting in stabilization of target proteins. 

DUBTACs consist of three components: 

• DUB recruiter
• Target protein binder
• Linker connecting both entities

DUBTACs restore protein levels, function, and rescue target proteins from degradation via the proteasome. 

Yes. Lifesensors has several years of experience in developing and validating custom assays for DUBs and E3 ligases. The assays can be used in high throughput screening to discover small molecule modulators.  

What is the ideal substrate for measuring DUB activity? 

The majority of the DUBs show activity using the Ub-Rh110 substrate. However, some DUBs prefer Di-Ubiquitin substrates. LifeSensors offers K6-DiUb, K11-DiUb, K48-DiUb and K63-DiUb TR-FRET substrates for testing DUB activity as well as chain selectivity of DUBs.  

Lifesensors offers two separate panels of DUBs for testing compound selectivity. DUB Panel I comprises of 10 DUBs and DUB Panel II contains 22 DUBs. DUB Panel I is ideal for initial testing of compounds. Testing can be done in a single concentration format or dose response format to obtain IC50s.  

Lifesensors utilizes its TUBE-based homogeneous TR-FRET assay to measure E3 ligase activity. Biotin-TUBEs bind to the fluorescently labeled polyubiquitin chains synthesized by the E3 ligase resulting in a TR-FRET signal. This assay can measure E3 auto-ubiquitination or substrate ubiquitination.    

Lifesensors offers three separate panels of E3s for testing compound selectivity. There are 5 E3s in Panel I, 10 E3s in Panel II and 30 E3s in Panel III. Initial selectivity testing can be done using Panel I which comprises of members from different classes of E3s. Testing can be done in a single concentration format or dose response format to obtain IC50s.  

We can receive compounds in powder form, as DMSO stocks or as aqueous solutions. Compounds can be shipped to us as individual vials or in 96-well or 384-well plates.  

We sell inhibitors for use as internal controls for several DUBs. 

The DUB and E3 assays require reducing agent for maintaining their catalytic activity.  We recommend adding 2mM beta-mercaptoethanol or 10mM DTT to the assay buffer and using this assay buffer to make enzyme and substrate dilutions as well as performing the assays.  E3 assays contain multiple components and care must be taken to ensure that all the necessary components like E1, E2, E3, Ubiquitin and ATP are included in the assay.  

We recommend 50 mM HEPES, 100 mM NaCl, and 5 mM DTT, pH = 8.0. DTT ensures that the active site cysteine of the DUB is reduced, allowing DUBs to perform their best. When screening compounds in the assay, the addition of detergents such as CHAPS at 0.05% and Bovine Serum Albumin (BSA) at 0.01% can improve the assay performance and the quality of hits. Assay buffers containing 50mM Bicine or 50 mM Tris, pH = 8.0, can also be used.  1-2mM β-mercaptoethanol can also be used as reducing agent instead of DTT. Although these recommended assay buffers should yield the best results for cleavage of polyubiquitin chains with most DUBs, certain DUBs may require additional buffer optimization and/or cofactors/binding partners for best performance.

We keep all of our chains at -80°C here at Lifesensors, but they are safe to thaw on ice. Do not leave them out for extended periods of time and always return them to -80°C when finished for the day.

Depending on the activity of the DUB and which DUBs you are using, you can get complete or partial cleavage. Always include a “-DUB” control to see what the chain looks like before cleavage.  If you cleave a tetra-ubiquitin, for example, then you should end up with a gel that has a band at Tetra-, Tri-, Di-, and monoubiquitin. This means the DUB successfully cleaved the ubiquitin. If you see only monoubiquitin and you started with a tetra-ubiquitin, then you had 100% cleavage.

If you are just running a Western blot, we do not recommend loading more than 50 ng at a time as overloading could yield “smudged” Western Blot results.

No, freeze thaws should not affect any chain ubiquitin. Freeze thaws will, however, affect the DUBs used to cleave them as they are enzymes. The DUBs will begin to lose activity after multiple freeze thaws. If you are worried about your chains or your DUBs, aliquot your desired amounts and thaw as needed.

Non-Cleavable DUB-Resistant Ubiquitin Chains are linear, recombinant proteins expressed and purified from E.Coli. They are modified to be resistant to even the most promiscuous DUBs. When incubated with a high DUB concentration, normal chains show almost 100% cleavage, while DUB-Resistant Chains exhibit little to no cleavage at 100 nM DUB concentration.

Linear ubiquitin chains are recombinantly expressed and purified from cells. Linear chains do not have specific linkages unlike non-linear chains. Any K6, K11, K27, K29, K33, K48, and K63 linked Di, Tri, and Tetra Ub chains are made enzymatically in vitro. We use a combination of chain specific E2s and DUBs to first assemble the chains and then remove unwanted chains before purifying the specific chains.

K to R mutants are ubiquitin mutants that have their individual lysines at K6, K11, K27, K29, K33, K48, K63, or all (K0) lysines mutated to arginine (R). This is to generate different types of ubiquitin chains depending on the end user’s goals.

Ubiquitin comprises 7 lysine residues among its 76 amino acids. The attachment of ubiquitin to specific lysine residues in proteins, including itself, is crucial for creating diverse substrate-ubiquitin structures. This versatility allows the pathway to target proteins for various fates, such as degradation or control of specific cell cycle phases. For instance, K48 chains promotes proteasomal degradation, K11 regulates substrates in the anaphase-promoting complex, and K63-linked ubiquitination controls the interaction, translocation, and activation of proteins.

Ubiquitin Rhodamine and AMC are ubiquitin derivatives used as substrates to test the activity of DUBs. When the DUB cleaves Ubiquitin from Ub-Rhodamine or UbAMC, the release of free Rhodamine and AMC results in an increase in fluorescence. These substrates allow for simple, fast, quantitative, and real-time monitoring of DUB activity.

K48 Tetra Ub linked Rhodamine is the same as above but meant for testing DUBs that are specific to K48 chains.

Tandem Ubiquitin Binding Entities (TUBEs) are industry-leading tools to detect and enrich/purify poly-ubiquitylated proteins from cell lines, tissues, and organs. Pairing this sample processing step with LC/MS analysis, generates a reproducible survey of the ubiquitome. We have worked with clients to investigate ubiquitin levels as a response to drug dosing, genetic knockouts, disease states, and others.

Clients receive their processed data in an excel file, as well as a summary report. We include graphical representations of ubiquitylation tends of your proteins. You will receive professional support when analyzing your data. See example report.

• Written report with summary of the TUBE based mass spec proteomics data, with sample prep protocols and LC-MS methods implemented for given samples along with sample description.
• Total Ubiquitinated identified will be presented in a list, as well as being organized on a distribution chart to show proportions.
• Total peptides identified will be presented in a list with length and mass information.
• Additional bioinformatic analysis for getting functional perspective of Proteomics data like DE analysis, cluster analysis and pathway & network analysis are available upon request at addition costs.

Your job ends at the shipment of frozen cells or tissue. Our scientists lyse your cells with a cocktail of inhibitors to protect the ubiquitylated species. The samples are then prepared using the TUBEs to pulldown the ubiquitylated proteins. Pan selective TUBEs are recommended for a global view of the ubiquitiome, but we also offer K48 and K63 linkage-specific TUBEs for specialized studies. Proteins are then separated on a gel by SDS-PAGE and are stained for analysis. After data is collected by LC-MS/MS we begin data analysis by running bioinformatic searches. The depth of our analysis depends on your needs and budget and we are happy to work with you to decide the best methods.

The controls for each experiment depend on the question being asked, but there are some guidelines that you might find useful. If you are looking into the effects of a drug or other compound, we recommend submitting a negative control. For genetic studies the negative control may be a wildtype sample. The use of inhibitors is another important control in this type of study. We recommend submitting samples treated with proteasome inhibitors, such as MG-132 to accumulate ubiquitylated proteins. We are happy to help you design a robust experiment around your specific research question, please contact us.

A protein thermal shift assay, sometimes called a thermoflour assay, measures changes in the thermal denaturation temperature (ΔTm, °C) of a protein resulting from the binding of small molecules. The assay is rapid and can be used for high throughput screening to identify and characterize small molecule compounds that bind to proteins. 

When a protein is gradually heated, the melting/denaturation of the protein exposes hydrophobic regions. In the presence of a dye (e.g. SYPRO Orange) the hydrophobic regions will bind to the dye, resulting in a pronounced increase in its fluorescence intensity. The temperature at which a protein is 50% denatured is called the melting temperature (Tm) of the protein. Binding of small molecules can either increase or decrease the Tm of the protein; we are measuring the change in the melting temperature relative to untreated protein.   

Typically, 1-5µM of target protein is sufficient to generate strong signal in the thermal shift assay.  However, the optimal concentration for individual proteins should be experimentally determined prior to any screening or compound testing.  

A thermal cycler that can hold 384-well plates and measure fluorescence is needed for thermal shift assay based high-throughput screening.  

While thermal shift assay should work with most proteins, some proteins may not be suitable for thermal shift assay. Smaller proteins with very low hydrophobicity or proteins that are not folded/aggregated are not suitable for thermal shift.   

SYPRO Orange is dissolved in DMSO. 

Surface Plasmon Resonance (SPR) is an optical technique for detecting the interaction of two different molecules. One molecule (the ligand) is immobilized on a gold-coated surface, while another molecule (the analyte) is injected over the surface. The technique measures a change in refractive index at the surface, which is directly proportional to the change in mass. Both protein-protein and protein/small molecule interactions can be investigated with this method. Binding response units (RUs) are measured in real-time and analyzed to determine kinetic parameters including the binding affinity (KD). 

We routinely use amine-coupling to immobilize proteins on to SPR sensor chips. We also use high affinity nickel chips to immobilize proteins containing histidine tags and amine-coupling of NeutrAvidin to capture biotinylated ligands. 

For small molecule binding studies, we recommend the target protein to have Avi-Tag. Biotinylated proteins allow rapid immobilization using Neutravidin sensors and are ideal for small molecule binding studies.  

For immobilization you only need ~25-50ug of ligand in a suitable buffer. If using amine coupling the buffer should not contain free amines (e.g. TRIS buffer is not suitable). The stock ligand concentration should be sufficiently high (>0.5mg/ml) because the ligand will be diluted in immobilization buffer. 

It depends on the binding kinetics between ligand and analyte. For kinetic studies, a range of concentrations between 0.01-100 times the KD is injected.  

We use a Reichert SPR4 instrument which supports a four-channel flow cell. This allows up to 3 experimental channels with one reference channel. The 3 experimental channels are isolated from each other, allowing for up to three different ligands to be screened. Data is processed using TraceDrawer. 

Depending on the immobilization protocol, about 45-90 minutes total is needed to prepare a chip. When the immobilization conditions are known, a simple amine coupling can be done within 30 minutes. 

The amount of ligand depends on the application. Protein-protein interactions typically require less surface density of ligand (ca. 1000-4000 RU). To measure small molecule (MW >300 Da) binding to proteins, one typically wants a saturated ligand surface; ~8000-10,000 RU of captured target protein ligand should be sufficient.  

For the standard amine-coupling, 10mM sodium acetate (pH 4.0-5.5) buffer is used. Ligand storage buffer should not contain free amines (e.g. TRIS). 

Regeneration buffers often need to be screened depending on the type of SPR experiments and interactions measured. Some typical regeneration solutions include 0.5-1M NaCl (ionic), 50 mM Glycine (pH 3.0 or 9.5), ethylene glycol (hydrophobic), or some combination thereof. 

Yes. SPR can reliably determine the binding affinity KD for small molecules and target proteins. Typically, the target protein of interest is captured onto the sensor chip and a series of compound doses are injected to generate dose-response binding curves.