FAQs – Research

The ExoCap™ kit isolates intact extracellular vesicles, specifically by targeting antigens associated with exosomes and has been optimized for serum and plasma samples.

ExoCap™ uses functionalized Magnosphere™ magnetic particles for exosome immunoprecipitation.  These beads are coupled to antibodies that recognize the exosome surface antigens (CD9, CD63, CD81, or EpCAM). After adding Treatment Buffer and incubating the sample with the ExoCap™ capture beads, the tube is placed on a magnetic tube stand and the supernatant is removed, leaving intact exosomes attached to the ExoCap™ capture beads as an exosome-based complex.

ExoCap™ enables simple, high purity capture of exosomes in as little as 20 minutes up to 24 hours. The kit does not require messy precipitation or tedious ultracentrifugation. Captured exosomes are easy to manipulate as Magnosphere™ beads are visible and respond quickly to magnets.

The ExoCap™ kit consists of magnetic particles coupled to antibodies that recognize the exosome surface antigens (CD9, CD63, CD81, or EpCAM), treatment buffer, washing/dilution buffer, and exosome elution buffer. ExoCap™ uses functionalized Magnosphere™ magnetic microparticles for exosome separation. These beads are coated with a JSR Life Sciences’ proprietary polymer and cofactors which specifically bind to exosomes.

There are 5 ExoCap™ kits available:  ExoCap™ CD9 Capture Kit, ExoCap™ CD63 Capture Kit, ExoCap™ CD81 Capture Kit, ExoCap™ EpCAM Capture Kit, and ExoCap™ Composite Kit. The composite kit is a combination of the 4 capture beads mixed together. Each kit differs by the targeted antigen. The composite kit is capable of capturing all four antigens (CD9, CD63, CD81 and EpCAM).

If researcher is starting immunoprecipitation studies without previous exosome characterization, we recommend beginning with the Composite Kit to perform preliminary characterization studies. If the exosome of interest are found to have a predominant population of CD9, CD63, CD81, or EpCAM exosomes then use of their corresponding capture kit will be beneficial.

A magnetic stand designed to hold 1.5 or 2.0 mL tubes is required for use with the ExoCap™ kit. This stand is generally sold by third party vendors and can hold a maximum of 16 tubes.

The beads are single-use only. Eluting exosomes from the beads using Lysis buffer for SDS-PAGE, such as Laemmli buffer, or Exosome Elution Buffer, denature the antibody on the beads so that the activity of the ExoCap™ is diminished.

ExoCap™ is designed to capture exosomes based on immunoaffinity to the targeted surface antigens. Due to exosome heterogeneity in plasma exosomes, there may be exosomes without one of the four antigens we are targeting. These exosomes will not be isolated by the ExoCap™-SP kits. There is an ExoCap™ Streptavidin kit available which will capture exosomes based on your biotinylated antibody of interest.

The kit does not contain bovine serum albumin. This kit was manufactured without any animal components.

The elution buffer is a proprietary compound which contains a denaturing agent. The Washing/Dilution Buffer is a proprietary compound which contains a surfactant.

The elution buffer is a proprietary compound which contains a denaturing agent. The Washing/Dilution Buffer is a proprietary compound which contains a surfactant.

We have seen a limited amount of antibody leaching with the Exosome Elution Buffer. If the leached antibody from the beads makes it difficult to identify the protein of interest on western blot analysis, please try to use detection antibody which does not recognize denatured mouse lgG.

Yes. Adding reducing agents will cause some antibodies to come off the beads. To avoid the leaching H chain and L chain of antibody from the beads, please use reducing agent if necessary after getting rid of the beads from the eluted solution.

No, plasma and serum samples can proceed directly in the ExoCap™ kit protocol.

The beads would tend to aggregate if there is an excess amount of calcium in the sample.

If the sample has been pre-concentrated, we recommend titrating the bead amount based on target abundance.

ExoCap™-SP kits have been optimized for serum and plasma, but these kits can also be used for exosome-conditioned sample media.

Downstream applications such as Nanosight which measure exosome size and concentration will require an elution step.

The captured exosomes can be used directly for downstream applications such as western blot analysis or RT-PCR without an elution step.

The Exosome Elution Buffer interferes with mass spectrophotometry. An immediate buffer exchange into a non-denaturing buffer is recommended.

The ExoCap™ kit should be stored at 4ºC.

We recommend that the buffers come to room temperature prior to performing the immunocapture. To avoid unnecessary changes in temperature, the elution buffer can remain stored at 4ºC if intact exosomes are not required in the downstream process.

No. Freeze-thaw cycles will cause denaturation of the antibodies.

The ExoCap™ kit isolates intact extracellular vesicles using biotinylated molecules, such as anitbodies to exosome surface marker proteins.

Exosomes are cell-derived vesicles originating from multi-vesicular bodies and found in biological fluids including blood, urine, and cell culture mediums. Sizes of these extracellular vesicles range between 20-100nm. Exosomes play a key role in cell-cell communication and have been implicated as a disease biomarker for cancers and immune system disorders.

The ExoCap™ Streptavidin kit uses functionalized Magnosphere™ magnetic particles for exosome separation, which is coated with JSR Life Sciences’ proprietary hydrophilic polymer to decrease non-specific binding. These beads able to be coupled with a researcher’s biotinylated molecules against exosome surface marker proteins. After incubating the sample with the ExoCap™ Streptavidin beads, the tube is placed in a magnetic tube stand and the supernatant is removed, leaving intact exosomes attached to the ExoCap™ Streptavidin beads as an exosome-bead complex.

The ExoCap™ Streptavidin kit enables easy, high purity capture of exosomes in as little as 20 minutes up to 24 hours. The kit does not require messy precipitation or tedious ultracentrifugation. Captured exosomes are easy to manipulate as Magnosphere™ beads are visible and respond quickly to magnets.

The ExoCapTM Streptavidin kit consists of Streptavidin magnetic particles, treatment buffer, and washing/dilution buffer. The ExoCapTM Streptavidin kit uses functionalized MagnosphereTM magnetic microparticles for exosome separation. These beads are coated with a JSR Life Sciences’ proprietary polymer and streptavidin which specifically associate with biotinylated molecules.

A magnetic stand designed to hold 1.5 or 2.0 mL tubes is required for use with the ExoCap™ Streptavidin kit. This stand is generally sold by third party vendors and can hold a maximum of 16 tubes.

The beads are single-use only. Eluting exosomes from the beads using Lysis buffer for SDS-PAGE, such as Laemmli buffer, or Exosome Elution Buffer, denature the antibody on the beads so that the activity of the ExoCap™ is diminished.

The kit does not contain bovine serum albumin. This kit was manufactured without any animal components.

The Washing/Dilution Buffer is a propietary compound which contains a surfactant.

Yes. Adding reducing agents will cause some antibodies to come off the beads. To avoid the leaching H chain and L chain of antibody from the beads, please use reducing agent if necessary after getting rid of the beads from the eluted solution.

No, the pre-cleared samples can proceed directly into the ExoCap™ Streptavidin kit protocol.

No answer yet on website.

For cell culture media, we recommend 100µL of beads for sample volumes between 100µL and 1000µL.

If the sample has been pre-concentrated, we recommend titrating the bead amount based on the target abundance.

The ExoCap™ kit should be stored at 4ºC.

We recommend that the buffers come to room temperature prior to performing the immunocapture. To avoid unnecessary changes in temperature, the elution buffer can remain stored at 4ºC if intact exosomes are not required in the downstream process.

No. Freeze-thaw cycles will cause denaturation of the antibodies.

We obtain serum-starved NRK cells as follows:

1. Exchange culture medium with Hanks’ Balanced Salt Solution (serum-free).
2. Incubate NRK cells in the Hanks’ Balanced Salt Solution (serum-free) for 2-4 hours.

Starvation can also be induced using serum-free DMEM (Dulbecco’s modified Eagle’s medium), but amino acids in the DMEM reduce the level of starvation.

Please note that optimal conditions may differ depending on the cell line, so optimal experimental conditions should be determined by end users.

We recommend 15% gel. When using a 10% gel, the bands of LC3-I and LC3-II overlap each other, making it difficult to distinguish these two bands.

Please follow the protocol in the data sheet and check the points listed below.  Make sure the sample buffer contains SDS. We recommend the use of SDS-PAGE sample buffer (Laemmli’s sample buffer).

When you detect LC3 with monoclonal antibodies, the washing step after blocking is necessary. Wash with 0.05% Tween-20/PBS (5 mins x 3 times) to produce a stronger LC3-II signal.  Cell suspension of Human LC3B transfected cells is available from MBL as a positive control in WB.

Please refer to the following research article.

Interpretation of LC3 in Western Blotting is described in detail. Mizushima N, Yoshimori T. How to interpret LC3 immunoblotting. Autophagy. 3(6): 542-545, 2007 PMID:17611390

Klionsky DJ, et al., Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes, Autophagy 4(2), 151 (2008) PMID: 18188003

Klionsky DJ, et al., Guidelines for the use and interpretation of assays for monitoring autophagy, Autophagy 8(4), 445 (2012) PMID: 22781101

We use Digitonin (Sigma, D141) in PBS for permeabilization and make a fresh preparation at a final concentration of 100 μg/mL. Triton X-100 is not suitable as a permeabilization solution for immunocytochemistry.

We use 4% PFA/PBS. Methanol fixation or acetone fixation are not recommended.

Please use 10% formalin solution (3.7% Formaldehyde) or 4% PFA/PBS.

The use on the cryosections has not been tested by MBL at this time.

Different antibodies are recommended depend on applications. Please check the information below.

WB:code no. M186-3, PM036

IP:code no. M152-3, PM036

IC:code no. M152-3, PM036

FCM:code no. M152-3, PM036

IHC:code no. PM036

MaxBlot is a reagent that enhances the signal in WB and ELISA.

It is used as an antibody diluent. The MaxBlot stock solution is used to make dilutions of antibodies.

The buffer components have been optimized to enhance the S/N ratio.

Solution 1: The solution has been prepared to enhance the binding affinity of antibodies.
Solution 2: The binding affinity of antibodies is enhanced at the same time that non-specific reactivity is suppressed.

The salt concentration is 50mM and the pH is 7.2-7.4 in both Solution 1 and Solution 2.  The formulation of the components is different for each solution.

Solution 1: For dilution of primary antibodies

Solution 2: For dilution of secondary antibodies There are cases where it is also effective to use Solution 2 to make dilutions of primary antibodies.

It depends on the antibody. Therefore, it is suggested that a study be made for each experiment.

No, MaxBlot cannot be used as a blocking reagent. No special blocking reagent for MaxBlot is provided. Since the optimal blocking reagent depends on the antigen or antibody, it is necessary to make a thorough study of the conditions where block is performed before an antibody reaction.

1% Skim milk/PBS is used in WB and 1% BSA/PBS is used in ELISA in our internal studies.

HRP-labeled antibody and AP-labeled antibody can be used.

Although we have not made internal studies, it is considered that if the antibody is one that can be stored as a diluted solution in ordinary antibody diluent buffer, it can be used by a similar method of storage.  Ultimately, the researcher will need to make study in advance.

Chemiluminescent and color development methods can be used.

In the case of WB, high-sensitivity luminescent reagents can be used.

For the recovery of small RNAs, RIP-Assay Kit for microRNA should be used.

RIP-Assay Kit is designed to recover large RNAs but not small RNAs.

RIP-Assay Kit for microRNA is designed to recover both small RNAs and large RNAs. Moreover, by following different procedures, small RNAs and large RNAs can be isolated separately, or only small RNAs can be recovered. By recovering small RNAs which bind to RBPs/large RNAs, RIP-Assay Kit for microRNA enables you to study how the intracellular event of your interest is regulated by not only RBPs but also small RNAs. The kit is also useful for the identification of target RNAs that specifically bind to miRNAs of your interest.

We recommend preparing 4×10^6 – 2×10^7 cells. However, appropriate cell number is different depending on the cell lines.

For the first time users, it is recommended to use 1×10^7 cells/sample and optimize the cell number depending on the experimental conditions.

If the contamination of large RNAs does not affect the following experiments, 2-step method is appropriate, since the recovery rates are high for both small RNAs and large RNAs. If the contamination of large RNAs should be avoided, isolate small RNAs by Separation method or 1-step method.

To evaluate quality and quantity of obtained miRNAs, we recommend visualization of small RNAs using GelStar or silver staining following RNA electrophoresis. After visualization, you can check the size, quantity and quality of obtained miRNAs compared to RNA ladder and control miRNAs. RNA quantitation reagents are also available if the obtained miRNAs are in the measurement range of the reagents.

For downstream analysis of the obtained RNAs, we recommend the followings.

• Northern blot or RT-PCR for specific miRNAs
• RT-PCR, microarray or sequencing to identify miRNAs

Depending on the following analysis, suitable isolation methods are different. For microarray analysis, the eluate can be used without any purification. For cloning or sequencing, miRNAs are recovered after the adapter ligation to miRNAs on beads.

Kits for cloning small RNAs are also commercially available.

With RIP-Assay Kit for microRNA, both large RNAs and small RNAs can be recovered. With Bioanalyzer, we examine whether the large RNAs obtained by RIP are degraded or intact. By checking the wave patterns of each RBP, we also analyze whether the obtained RNAs are specific to each RBP.

Recovery of small RNAs is examined by silver staining, since small RNAs are undetectable by NanoDrop or Bioanalyzer.

Protease inhibitors are added for the purpose of avoiding degradation of RBPs and antibodies, but not for the stabilization of RNA-RBP interactions. When you are planning to recover RNAs having only weak interaction with RBPs, cross-linking is necessary.

We do not examine the influence of formaldehyde cross-linking. By using formaldehyde, protein-protein interactions as well as nucleic acid-protein interactions are cross-linked. Therefore, we think formalin is not very suitable for RIP-Assay after cross-linking.

A licensing agreement is required to use the RIP-Assay Kits and RIP-Chip Technology for business purposes. Please contact us for more information.

RNP* immunoprecipitation (RIP) is a procedure used in studying RNAs binding to RBPs (RNA Binding Protein) by isolating the RNPs through immunoprecipitation using specific antibodies against RBPs.

*RNP is a complex formed between RNA and RBP in a cell.

It is expected that the” RNAs directly binding RBPs” and  “RNAs indirectly binding RBPs
such as RNA interacting with other RBP formed complex with target RBP” in the sample become concentrated. When comparing the rRNA and the tRNA in the sample after RIP with mRNA (and miRNA) which was initially just a small percentage of the total RNA, we find out that the concentration has increased. In addition, analysis for the enriched RNAs are low background and able to easily find out their relationship (for example, working at same pathway).

Quantitative

• Measuring the quantity of the UV (A260nm) absorbed (We measures by NanoDrop)
  However, this method cannot be used on low concentration samples due to low sensitivity.

Qualitative

• Waveform analysis using an Agilent Bioanalyzer
  It is possible to test with high sensitivity the size distribution of the nucleic acids in a RIP sample (capillary electrophoresis).
• Sequence determination using sequencing (classical sequencing)
  The base sequence of the RBPs can be determined.
• Real-time quantitative PCR analysis
  Specific detection and semi-quantitative analysis.
• Microarray Analysis
  A filter is available to narrow down analysis results from tens of thousands of mRNA.
• High-throughput sequencing
  The base sequence of the RBPs can be determined.

The RIP ability differs depending on the antibodies used. In addition, it sometimes happens that RNAs are not isolated even RBPs are precipitated.
Hence we recommend using purified antibody and checking if enough quantity/quality of RNA is isolated and addition of High-Salt Solution is necessary or not before doing actual experiment.

The RIP-Assay Kit is intended for analyzing the “RNP in the cytoplasm”. Lysis buffer provided RIP-Assay Kit does not completely solublize nuclear membrane, hence nuclear RNPs can be hardly collected.

If you do not perform RIP-Assay without cross-linking, using RiboTrap Kit and RIP-Assay Kit for microRNA will enable RIP-Assay for nuclear RNPs. Please prepare nuclear lysate according to the procedure of RiboTrap Kit, and then perform RIP-Assay. In this case, it’s better to optimize sonic condition to reduce DNA contamination without RNA degradation.

It differs depending on the cell but please prepare a sample of 4×10⁶-2×10⁷ cells. Initially, we recommend considering a 1×10⁷cells/sample

Dynabeads^® (Invitrogen) can be used for Protein A and Protein G. You can also use Magnosphere™ MS300 (JSR).

In some case, the background may be increased depending on how hard the cross-linking is when using agarose beads.
We recommend reagents such as the listed below;

• Protein A (or G) Sepharose CL-4B (GE Healthcare)
• Immobilized Protein G Plus (Pierce; code. 22852)

RIP-Assay Kit does not support cross-linking . You will need some protocol modifications.

The addition of protease inhibitors is effective for preventing degradation of the antibodies and the RBPs rather than stabilizing interaction between RNAs and RBPs. You will need cross-linking if you want to collect RNAs weakly bound to RBPs.

We have not examined cross-linking in formalin. In formalin, not only the nucleic acid and protein, but also between proteins are cross-linked. Therefore, we think formalin is not very suitable for RIP-Assay after cross-linking

A license is required to use the RIP-Assay Kit and RIP-Chip Technology for business purposes. Please contact us for more information.

To compare the target RNA with negative control, you will use equal amounts of RNAs. To compare the target RNA with total RNA (RIP RNA vs toal RNA), you will use equal volume of RNAs. Please see the following example:

Example
If you have the following three as a sample and use 1 ng as a template for RT-PCR:

1. Control IgG, (concentration: 2 ng/μL)
2. RBP-RIP RNA, (concentration: 20 ng/μL)
3. Total RNA, (concentration: 100 ng/μL)

You will use each 1 ng of sample 2 and sample 3. To compare sample 1 with sample 2, you will use 1/20 μL of sample 2 and 1/20 μL of sample 1.

RIP-Assay Kit cannot collect small RNAs. RIP-Assay Kit for microRNA can collect both small and large RNAs. Furthermore, it can also collect small and large RNAs separately as well as collect only small RNAs. By collecting small RNAs bound to RBPs/large RNAs, you will able to study how your interesting intracellular event is regulated by not only RBPs but also small RNAs. In addition, it is also useful for the identification of target RNAs specific binding to your interested miRNAs.

In microarray, most of RNA expression patterns do not change but a part of them drastically changes. Hence it makes possible to normalize and analyze array data. However, in RIP-Chip, the comparison target RBP-RIP with negative control or other RBP-RIP is nonsense bacause the RNA population is mostly different from each other. Microarray is good for comparison of different two conditions such as drug-treated or not, and gene transfectant or mock.

Yes. It can be used for the liver tissue sample.

RiboTrap is an immunoaffinity method to isolate RNA-binding proteins by using RNA of your interest as bait. This method enables to isolate unknown proteins as well as known proteins.

RiboTrap is used to identify proteins that bind to a specific RNA. It enables to identify unknown proteins as well as known proteins. EMSA is used to detect the binding between specific sequences of nucleic acids and proteins 

We synthesize BrU-labeled RNA by adding BrUTP to the reactive substrate of in vitro transcription. The recommended molar ratio of BrUTP to standard UTP is 1:1 to 1:3. To determine the appropriate ratio, uracil content in the RNA sequence should be considered.

The incorporation rate of BrUTP depends on the uracil content of the RNA sequence and BrUTP-adding ratio. Moreover, BrUTPs are randomly incorporated into the RNA when performing in vitrotranscription. We recommend using synthesized oligonucleotides as bait if you need BrU labeling at the specific positions in the RNA.

According to our in-company data, we think that the possibility is low.

We conclude that small ncRNAs can be used for RiboTrap. The BrU-labeled positions and numbers in the target sequence should be considered when performing RiboTrap using short-length RNAs. Synthesized RNA oligos can be used as bait if necessary.

Unincorporated BrUTPs inhibit the interaction between BrU-RNAs and antibodies via competitive binding. Thus, synthesized BrU-RNA should be purified after in vitro transcription.

We have not adjust the amount of protein, but we start experiments with 7-10×10⁷ cells.

• Wash Buffer I: The buffer with the mildest conditions. It allows the isolation of weakly-bound proteins as well as tightly-bound proteins. On the other hand, the mild condition gives higher background than Wash Buffer II and III.
• Wash Buffer II: The buffer with high ionic strength. It is suitable to isolate the proteins that tightly bind to RNAs.
• Wash Buffer III: The buffer with high detergency. It reduces proteins that indirectly bind to the target RNA.

Please select the wash buffer best suited for your purpose

We have not tried to use tRNA as blocking. Nonspecific binding can be avoided by washing with Wash Buffer included in RiboTrap Kit. For optimization of washing condition, we recommend using Wash Buffer I for primary screening and then using Wash Buffer II or III if needed.

RNase inhibitor is added in the Lysis Buffer. In RiboTrap as well as RIP-assays, please take all possible measures to avoid RNA degradation such as using nuclease-free grade reagents and instruments, wearing masks and gloves, and controlling temperature of the experiment environment.

We recommend following in vitro transcription kits;

• Riboprobe^® System-T7 (Promega; code. P1440)
• TranscriptAid™ T7 High Yield Transcription Kit (Thermo Fisher Scientific; code. K0441)

We recommend Immobilized Protein G Plus (Thermo Fisher Scientific; code. 22852). Dynabeads^®(Invitrogen; code. 10003D) and Protein G-Magnetic beads (MBL; code. MJS002V2) also can be used in RiboTrap experiments

We have not tried to isolate proteins binding to lncRNA with RiboTrap. However, it is possible by using several truncated forms of the RNA as bait.

The isolation of miRNA is possible. After the formation of antibody-bait RNA-RNP complex and washing steps, we have isolated miRNAs by Separation method of RNA extraction protocol in RIP-Assay Kit for microRNA. The isolated miRNAs were then cloned and sequenced. Depending on the RNA of your interest, amounts of isolated miRNAs may be small compared to the RIP-Assay with antibodies against RISC components. Therefore, we recommend scaling up the experiment.