FAQs – Research

General

What are the warranties?

MBL International Corporation guarantees the material to be of merchantable quality at the time of shipment. Our liability is limited only to replacement of products up to the amount of the purchase price. MBL makes no other warranty, expressed or implied, including any implied warranty of fitness for a particular purpose or of merchantability. In no case shall MBL assume any responsibility for the use or handling of the product or any liability for consequential, special, indirect, or incidental damages resulting there from.

What are the payment terms?

Payment terms are net 30 days from the invoice date. Prices are FOB Woburn, Massachusetts or F.O.B. Des Plaines, IL. (see shipping information above). All prices quoted are in U.S. dollars. Prices are subject to change without notice. For customer convenience, MBL International Corporation will accept VISA, Master Card or American Express for purchases. Credit card approval must be obtained prior to shipment.

Why can't I see any prices?

Our prices can be seen by all our registered US customers. If you are a US customer and cannot see the prices, please Login. If you are not a US customer, please click on the Distributor link in the contact area to find your local Distributor.

Can I modify the assay protocol?

The protocol for each product should be followed exactly to ensure that the product performs to its specifications. MBLI products are designed to be optimized for the highest possible accuracy and sensitivity.

Can I mix reagents from different ELISA kits?

This is not recommended. The reagents provided are always specific to a particular analyte and kit lot. Please contact our Technical Support for more information

ExoCap-SP

What is the ExoCap™-SP kit used for?

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

What are exosomes?

Exosomes are cell-derived vesicles originating fro em 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.

How are exosomes captured by the ExoCap™ kit? How does it compare to other exosome capture techniques?

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.

What is the ExoCap™ kit made of?

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.

What types of ExoCap™ kits are available? How are they different? How do I know which kit is best for me?

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.

Are there special instruments required to use the ExoCap kit?

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.

Can ExoCap™ capture beads be reused?

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.

Are all exosomes being captured from the sample?

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.

Does the kit contain BSA?

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

What is in the Exosome Elution Buffer? What is in the Washing/Dilution Buffer?

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

Do antibodies come off the beads after eluting exosomes with the Exosome Elution Buffer?

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.

Do antibodies come off the beads after adding reducing reagents?

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.

Is there a precipitation step needed for sample prep?

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

Why are my beads aggregating?

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

Do I need more beads if I am using more sample?

Start with the recommended amounts of beads and sample + Treatment Buffer volume based on the downstream assay. If any adjustment is needed, adjust either sample volume + Treatment Buffer or bead amount but not both at the same time.

Will adding more beads reduce capture efficiency?

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

What sample media can ExoCap™ be used with?

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

What are some downstream applications that require the elution step?

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

Is it necessary to elute the exosomes for downstream applications such as western blot analysis or RT-PCR?

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

Does the Exosome Elution Buffer interfere with mass spectrophotometry?

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

How should ExoCap™ kit be stored?

The ExoCap™ kit should be stored at 4ºC.

Do I need to let ExoCap™ buffers come to room temperature before use?

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.

Can ExoCap™ be stored in a freezer?

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

ExoCap Streptavidin

What is the ExoCap™ Streptavidin kit used for?

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

What are exosomes?

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.

How are exosomes captured by the ExoCap™ Streptavidin kit? How does it compare to other exosome capture techniques?

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.

What is the ExoCap™ Streptavidin kit made of?

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.

Are there special instruments required to use the ExoCap™ Streptavidin kit?

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.

Can ExoCap™ Streptavidin beads be reused?

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.

Does the kit contain BSA?

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

What is in the Washing/Dilution Buffer?

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

Do antibodies come off the beads after adding reducing reagents?

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.

Is there a precipitation step needed for sample prep?

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

Do I need to add more beads if I am using more sample?

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

Will adding excess beads reduce capture efficiency?

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

How should ExoCap™ kit be stored?

The ExoCap™ kit should be stored at 4ºC.

Do I need to let ExoCap™ buffers come to room temperature before use?

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.

Can ExoCap™ be stored in a freezer?

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

 

Autophagy

How can I perform serum starvation?

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.

What percentage of gel is suitable for detecting LC3 in Western Blotting?

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.

How can I detect LC3 in Western Blotting?

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.

Do you have any information of how to interpret LC3-I and LC3-II bands detected in Western Blotting?

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

What should I take notice of when I perform immunocytochemistry using Anti-LC3 antibodies?

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.

Which fixatives are recommended for immunocytochemistry (IC)?

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

Which fixatives are recommended for immunohistochemistry (IH)?

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

Can Anti-LC3 Antibodies by MBL be used for cryosections?

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

Which antibody is recommended?

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

What kind of reagent is MaxBlot?

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

How is MaxBlot used?

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

What is the principle of signal enhancement by MaxBlot?

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.

What are the components of MaxBlot?

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.

Is there information about the different uses of Solution 1 and Solution 2?

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.

For the case of a primary antibody with an enzyme gel, which solution should be used to make dilutions?

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

Is it possible to use MaxBlot as a blocking reagent?

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.

What is used as the blocking reagent for the data in the datasheet?

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

Is there information about enzyme labels which can be used?

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

Is there information about the storage of diluted antibodies?

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.

Is there information about suitable methods for detection?

Chemiluminescent and color development methods can be used.

How can the sensitivity be enhanced further?

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

RIP for microRNA

What is the difference between RIP-Assay Kit for microRNA and RIP-Assay Kit?

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.

How many cells are needed to obtain small RNAs contained in RNP complexes?

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.

What is the suitable RNA isolation method to obtain miRNAs most efficiently?

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

How can miRNAs obtained with RIP-Assay Kit for microRNA be evaluated?

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

Besides gel extraction, what method can be used to isolate 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.

Why do you perform both analyses with Bioanalyzer and silver staining to check the quality of recovered RNAs?

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.

Are RNAs stabilized sufficiently by adding protease inhibitors without cross-linking?

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.

Does formaldehyde (formalin) cross-linking affect the RNA isolation with RIP-Certified antibodies?

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.

Is a licensing agreement required to use RIP-Assay Kits and related technologies for commercial purposes?

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

RIP-Chip

What type of experimental procedure is RIP?

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.

What is peculiar about the samples obtained from the RIP procedure?

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).

What are the different methods used in analyzing the samples obtained for the RIP procedure?

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.

Can it be used in performing RIP-Assay using homemade antibodies?

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.

Can you carry out RIP-Assay using Tag antibodies?

We have confirmed that anti-Myc-tag antibody (M047-3 (clone: PL14)) from MBL can be used in RIP. Other antibodies have not been tested.

Can you isolate the RNP clusters in the nucleus?

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.

What about the fact that the RIP-Assay Kit is unsuitable for nuclear RNA isolation, are there any ways to resolve this?

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.

How many cells are needed for RIP-Assay?

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

Is it possible to use magnetic beads?

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

Is there any particular recommendation or warning when using beads that can be used with the RIP-Assay Kit?

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)

I would like to do RIP-Assay with cross-linking using RIP-Assay Kit. Is it possible?

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

Can the RNA be stabilized just by adding Protease inhibitors without carrying out cross-linking?

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.

Is RIP-Certified antibody useful for RIP-Assay with (formalin) cross-linked?

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

Is a license or some sort of contract required to use RIP-Assay Kit for business purposes?

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

When amplifying the target sequence by RT-PCR, how much RNA is required for reverse transcription after its purification?

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.

What is the difference between RIP-Assay Kit and RIP-Assay Kit for microRNA?

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.

I would like to compare RIP RNA with negative control in microarray. Is it possible?

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.

Can the RIP-Assay Kit be used for the liver tissue sample?

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

RiboTrap

What is RiboTrap?

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.

What is the difference between RiboTrap and EMSA (gel shift assay)?

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

How do you perform BrU-RNA synthesis?

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.

How much are BrUTPS incorporated into the RNA?

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.

Is there any possibility that BrU labeling inhibits the binding or RNAs and proteins?

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

Is it possible to isolate proteins binding to small ncRNA using RiboTrap?

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.

Is there any possibility that unincorporated BrUTPs after in vitro transcription inhibit the binding between antibody and BrU-RNA?

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

How much protein should be used in RiboTrap Kit?

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

What is the difference among three Wash Buffers?

  • 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

Have you added tRNA to reduce nonspecific binding?

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.

Is there any possibility that RNAs are degraded when mixing BrU-labeled RNAs and Cell Lysate?

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.

Are there any in vitro transcription kits that you recommend?

We recommend following in vitro transcription kits;

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

Are there any recommended beads suitable for RiboTrap Kit?

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

Is it possible to isolate proteins binding to long-length RNA such as Xist using RiboTrap?

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.

Is it possible to isolate miRNAs as well as proteins using RiboTrap Kit?

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.