Protein-Nucleic Acid Interactions Support—Troubleshooting

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DNA EMSA

Lightshift™ Chemiluminescent EMSA assay でバンドシフトが確認できなかった場合、どうすれば良いでしょうか？

Here are possible causes and solutions:

Cause

Solution

DNA/protein complex may have been disrupted by vortex mixing or heating

Try running the gel with cold buffer

Not enough extract was used

Use more extract

Extract degraded

Add protease inhibitors to the extract

System not optimized

System optimization can be achieved by using additives such as KCl, glycerol, MgCl₂ and/or detergents and determining their effects on the shift. Comparing the methods used in journal articles that successfully performed EMSA experiments with the proteins you are testing may help with optimization.

Lightshift™ Chemiluminescent EMSA Kit を使用しています。コールドプローブ（未標識プローブ）でバンドシフトのブロックが出来ないのはどうしてでしょうか？

The shift may be caused by non-specific binding or a DNA binding protein different than the one being tested.

Lightshift™ Chemiluminescent EMSA Kit を使用していますが、非特異バンドが検出されます。非特異バンドを無くすか減らすにはどうすれば良いでしょうか？

Non-specific bands may be reduced or eliminated by optimizing the binding reactions. Strong non-specific bands can often be mistaken for a positive result but will not be blocked by the addition of cold competitor probe. The following are examples of changes that may help reduce non-specific bands:

Reduce the amount of protein extract in the binding reaction.
Increase the amount of non-specific competitor DNA [i.e., poly(dI-dC)A·poly(dI-dC)].
Use a different non-specific competitor DNA [i.e., sonicated salmon sperm DNA or poly(dA-dT)A·poly(dA-dT)].
Preincubate the protein extract with non-specific competitor DNA before adding the biotinylated probe.
Shorten or redesign the probe used in the experiment.

32P を利用していた時はワークしていましたが、Lightshift™ Chemiluminescent EMSA Kit を使用するとシフトが確認されなくなりました。何が問題なのでしょうか？

The Lightshift™ Chemiluminescent EMSA Kit is used to detect a biotinylated probe in an EMSA. The binding conditions for each experiment must be optimized for the proteins tested. If an experiment was working when ³²P labeled probes were used, the same binding conditions from those previous experiments should be used, with the biotinylated probe to be substituted in place of the ³²P labeled probe. While commonly used reaction components are provided with the Lightshift™ Chemiluminescent EMSA Kit, these reagents may not be applicable in all situations.

Lightshift™ Chemiluminescent EMSA Kit を使用して検出したところバックグラウンドがかなり高く確認されました。原因として何が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Particulate in Blocking Buffer or Wash Buffer

Gently warm until no particulate remains

Contaminants in the TBE

Use high-quality reagents or filter TBE through a 0.2 μm filter before use

The transfer unit or sponges used were dirty

Use clean equipment and sponges that were not previously used for western blotting

ブロット上の斑点・スポットの原因は何でしょうか？

Speckling/spots can be caused by precipitate in the HRP conjugate or by air bubbles. Precipitate in the HRP conjugate can be removed by filtering the conjugate through a 0.2 μm filter or by centrifugation for 1 minute at maximum speed while air bubbles between the gel and the membrane should be removed before transfer.

Lightshift™ Chemiluminescent EMSA assay で非特異に DNA がシフトするのを防ぐにはどうすれば良いですか？

Use a non-specific competitor DNA such as poly(dI.dC).

Lightshift™ Chemiluminescent EMSA assay で検出シグナルが得られない場合や弱い場合があります。どうすれば良いですか？

Here are some suggestions to improve the results:

Make sure that the target DNA used is end-labeled with biotin.
Increase target DNA concentration.
Check integrity of target DNA to make sure it is not degraded.
Check the transfer protocol to make sure that the transfer is efficient.
Use the right type of membrane such as Biodyne™ B Nylon Membrane (Cat. No. 88518).
Cover the membrane completely during incubations to make sure it doesn’t dry out during the detection steps.
Make sure that the crosslinking is efficient.
Make sure that the 4X wash buffer is diluted to 1X.
Increase film exposure time during detection.

RNA EMSA

Lightshift™ Chemiluminescent RNA EMSA assay でシフトが確認されません。どうすればよいですか？

Here are possible causes and solutions:

Cause

Solution

RNA/protein complex may have been disrupted by vortex mixing or heating

Try running the gel with cold buffer

Not enough extract was used

Use more extract

Extract degraded

Add protease inhibitors to the extract

System not optimized

System optimization can be achieved by using additives such as KCl, glycerol, MgCl₂and/or detergents and determining their effects on the shift. Comparing the methods used in journal articles that successfully performed EMSA experiments with the proteins you are testing may help with optimization.

Lightshift™ Chemiluminescent RNA EMSA Kit を使用して検出したところバックグラウンドがかなり高く確認されました。原因として何が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Particulate in Blocking Buffer or Wash Buffer

Gently warm until no particulate remains

Excess free biotin in biotinylated RNA preparation

Remove excess biotin by extracting with chloroform or using a Sephadex™ Column

Excess biotin in extract preparation

Remove endogenous biotin using High-Capacity Streptavidin Agarose (Cat. No. 20357) to pre-clear extract

Contaminants in the TBE

Use high-quality reagents or filter TBE through a 0.2 μm filter before use

The transfer unit or sponges used were dirty

Use clean equipment and sponges that were not previously used for western blotting

Lightshift™ Chemiluminescent RNA EMSA Kit を使用して検出したところバックグラウンドがかなり高く確認されました。どういった理由が考えられますか？

Lightshift™ Chemiluminescent RNA EMSA assay で非特異に DNA がシフトするのを防ぐにはどうすれば良いですか？

Use a nonspecific competitor RNA such as tRNA or heparin.

Lightshift™ Chemiluminescent RNA EMSA assay で検出シグナルが得られない場合や弱い場合があります。どういった理由が考えられますか？

Here are some suggestions to improve the results:

Make sure that the target RNA used is end-labeled with biotin. Optimize labeling method and test for biotin labeling efficiency before assay.
Increase target RNA concentration.
Check integrity of target RNA to make sure it is not degraded.
Check the transfer protocol to make sure that the transfer is efficient.
Use the right type of membrane (Biodyne™ B Nylon Membrane, Cat. No. 88518).
Cover the membrane completely during incubations to make sure it doesn’t dry out during the detection steps.
Make sure that the crosslinking is efficient.
Make sure that the 4X wash buffer is diluted to 1X.
Increase film exposure time during detection.

RNA-Protein Pulldown

Magnetic RNA-Protein Pull-Down Assay で RNA の分解を防ぐにはどうすれば良いでしょうか？

Work in a clean nuclease-free environment.
Ensure that all plastics are nuclease-free.
After in vitro transcription, check RNA integrity by gel electrophoresis.

Magnetic RNA-Protein Pull-down Kit で最大どれくらいの長さのRNAを使用できるでしょうか？

RNA up to 450 nucleotides has been tested, but with optimization, labeling of longer RNA may be achievable. The RNA must have an accessible 3’-OH for the ligation reaction.

Magnetic RNA-Protein Pull-down Kit でラベル効率が低いです。ラベル効率をどう改善すれば良いでしょうか？

Ensure that the control reaction in the kit is run to confirm that the labeling reaction is efficient. If the control RNA labels well, but the test RNA does not, optimization is required. Addition of DMSO or heat may help to relax the structure. Additionally, ensure that there are no traces of organic reagent in the reaction (if RNA was extracted before labeling). Ensure that 70% ethanol wash was utilized after precipitation. Ensure that the RNA concentration is sufficient for the labeling reaction.

Magnetic RNA-Protein Pull-down Kit で RNA 結合タンパク質の回収効率が悪いです。何が起きたのでしょうか？

Here are possible causes and solutions:

Cause

Solution

Binding reaction was not optimized

Optimize incubation time, temperature, salt and detergent for binding reactions

Titrate amount of labeled RNA to protein lysate

Use a more concentrated lysate

Insufficient amount of magnetic beads used for capture

Increase amount of magnetic beads for capture

Insufficient amount of RNA used for capture

Increase amount of labeled RNA in reaction

Confirm good ligation efficiency

Magnetic RNA-Protein Pull-down Kit で RNA 結合タンパク質が全く回収できないです。どうすればよいですか？

Here are possible causes and solutions:

Cause

Solution

Insufficient amount of target protein in the sample

Increase amount of sample

Sample was not compatible with binding reaction

Buffer exchange sample using Zeba™ Desalting Columns

Binding reaction was not optimized

Optimize incubation time, temperature, salt and detergent for binding reactions

Titrate amount of labeled RNA to protein lysate

Use a more concentrated lysate

RNA binding protein had low affinity for labeled RNA

Add crosslinking reagent (e.g., UV, etc.) after protein has bound RNA

Magnetic RNA-Protein Pull-down Kit で非特異結合が多いです。どういった理由が考えられますか？

Here are possible causes and solutions:

Cause

Solution

Binding reaction was not optimized

Optimize incubation time, temperature, salt, and detergent for binding reactions

Insufficient washing stringency

Increase stringency of wash buffer; add salt and/or detergent

Ratio of labeled RNA to lysate was not optimized

Titrate labeled RNA with protein lysate

Reduce the concentration of lysate to ~2mg/mL

Magnetic RNA-Protein Pull-down Kit で RNA 結合タンパク質を回収後にウェスタンブロッティングを行うと検出シグナルが低いです。何が問題ですか？

Here are possible causes and solutions:

Cause

Solution

Insufficient signal

Increase amount of secondary antibody

Use a more sensitive chemiluminescent detection (e.g., SuperSignal Dura or SuperSignal Femto Chemiluminescent Substrate)

Poor antibody quality

Pre-screen antibody with cell lysate

Include cell lysate as a control on western blot

Protein was insufficient in lysate

Increase amount of sample

Identify alternate source of protein

Use a more concentrated lysate

Magnetic RNA-Protein Pull-down Kit で RNA 結合タンパク質を回収後に質量分析に掛けたいと考えています。どのようにすればできますか？

The elution fraction is compatible with preparation of peptides. Samples may be processed using the Mass Spec Sample Prep Kit for Cultured Cells (Cat. No. 89840). Alternatively, the elution fraction may be separated by denaturing PAGE. Bands of interest can be excised, and digested using the In-Gel Tryptic Digestion Kit (Cat. No. 89871).

Chromatin Immunoprecipitation

Agarose ChIP Kit（Cat. No. 26156）や Magnetic ChIP Kit（Cat. No. 26157）ではホルムアルデヒドで架橋を行う必要がありますか？

These kits were designed using formaldehyde as a crosslinker. The lysing, washing, and elution conditions have all been optimized for formaldehyde crosslinking. These steps are not optimal for native ChIP (no crosslinking,) or for other crosslinkers, such as EGS.

クロマチン免疫沈降とは何ですか？

Chromatin immunoprecipitation (ChIP) assays identify links between the genome and the proteome by monitoring transcription regulation through histone modification (epigenetics) or transcription factor:DNA binding interactions. The strength of ChIP assays is their ability to capture a snapshot of specific protein: DNA interactions occurring in a system and to quantitate the interactions using quantitative polymerase chain reaction (qPCR). Chromatin IP experiments require a variety of proteomics and molecular biology methods including crosslinking, cell lysis (protein-DNA extraction), nucleic acid shearing, antibody-based immunoprecipitation, DNA sample clean-up and PCR. Additional techniques such as gel electrophoresis are usually used during optimization experiments to validate specific steps. See here for more details.

Agarose ChIP Kit／Magnetic ChIP Kit 使用後のクロマチン断片が非常に大きい（> 1,000 bp）のですが、何が原因でしょうか？

Here are possible causes and solutions:

Cause

Solution

Crosslinking time was too long

Shorten crosslinking time

Cell to Micrococcal Nuclease (MNase) ratio was too high

Increase amount of MNase or decrease cell number (refer to the MNase digestion optimization protocol in Appendix A of the manual)

Agarose ChIP Kit／Magnetic ChIP Kit 使用後のクロマチン断片が非常に小さい（> 200 bp）のですが、何が原因でしょうか？

This indicates that the cell to Micrococcal Nuclease (MNase) ratio was too high. Decrease the amount of MNase or increase the cell number (refer to the MNase digestion optimization protocol in Appendix A of the manual).

Agarose ChIP Kit を使用したのですが核からクロマチンが回収できませんでした。何が起きたのですか？

This could be because your cell type requires more stringent handling to better lyse and release chromatin from the nucleus. Here are some suggestions:

Increase incubation with nuclei lysis buffer to 30 minutes and vortex for 30 seconds every 5 minutes.
Following the nuclear lysis step, sonicate the sample for 60 seconds using a 1/8 probe. Perform on wet ice in 3 pulses of 20 seconds with 30 second pauses between.
Following the nuclear lysis step, dounce the sample 20 times in a glass dounce homogenizer.

Magnetic ChIP Kit でコントロール（total input control）の PCR シグナルが出ないまたは低いのですが、何が原因でしょうか？

Here are possible causes and solutions:

Cause

Solution

PCR amplification conditions were not fully optimized

Optimize PCR conditions using samples known to contain the target amplicon

Check primer design

Insufficient amount of sample DNA added to the PCR reaction

Increase the amount of sample DNA added to the PCR reaction

Nuclei not fully lysed

Monitor sonication of nuclei by microscope to ensure full lysis

Agarose ChIP Kit でコントロール（total input control）の PCR シグナルが出ないまたは低いのですが、何が原因でしょうか？

Here are possible causes and solutions:

Cause

Solution

PCR amplification conditions were not fully optimized

Optimize PCR conditions using samples known to contain the target amplicon

Check primer design

Insufficient amount of sample DNA added to the PCR reaction

Increase the amount of sample DNA added to the PCR reaction

Note: Increasing the stringency of the nuclear lysis is not recommended unless there is no signal or low signal-to-noise in the IP. Excess sample can result in high background, decreasing the signal of the specific signal.

Magnetic ChIP Kit でポジティブコントロールサンプルの PCR シグナルが出ないまたは低いのですが、何が原因でしょうか？

Here are possible causes and solutions:

Cause

Solution

Insufficient chromatin amount in the IP reaction

Use at least 25 μg of chromatin for each IP

Insufficient antibody incubation time

Incubate antibody overnight

Nuclei not fully lysed

Monitor sonication of nuclei by microscope to ensure full lysis

Low-abundance target

Add more chromatin or magnetic beads (30μL)

Agarose ChIP Kit でポジティブコントロールサンプルの PCR シグナルが出ないまたは低いのですが、何が原因でしょうか？

Here are possible causes and solutions:

Cause	Solution
Insufficient chromatin amount in the IP reaction	Use at least 25 μg of chromatin for each IP
Insufficient antibody incubation time	Incubate antibody overnight
Incomplete elution from the Protein A/G agarose resin	Perform elution at 65°C and increase frequency of mixing

Magnetic ChIP Kit でポジティブコントロールサンプルとネガティブコントロールサンプルの PCR シグナルに差が見られない場合、何が原因でしょうか？

Here are possible causes and solutions:

Cause

Solution

Excess chromatin or antibody added to the IP

Add less chromatin or antibody

PCR amplification was measured outside the linear range of amplification

Decrease the number of amplification cycles used in the PCR reaction

Insufficient amount of sample DNA added to the PCR reaction

Add more sample DNA to the PCR reaction

Agarose ChIP Kit でポジティブコントロールサンプルとネガティブコントロールサンプルの PCR シグナルに差が見られない場合、何が原因でしょうか？

Here are possible causes and solutions:

Cause

Solution

Insufficient washing of the IP complex

Include an additional wash with Buffers 2 and 3

Excess chromatin or antibody added to the IP

Add less chromatin or antibody

PCR amplification was measured outside the linear range of amplification

Decrease the number of amplification cycles used in the PCR reaction

Magnetic ChIP Kit で検体の PCR シグナルが出ないまたは低いのですが、何が原因でしょうか？

Here are possible causes and solutions:

Cause

Solution

Insufficient amount of antibody added to the IP

Add more antibody to the IP

Antibody did not function in an IP

Verify that the antibody is qualified for ChIP or IP applications and has been handled and stored properly

Agarose ChIP Kit で検体の PCR シグナルが出ないまたは低いのですが、何が原因でしょうか？

Here are possible causes and solutions:

Cause

Solution

Insufficient amount of sample DNA added to the PCR reaction

Add more sample DNA to the PCR reaction

Insufficient amount of antibody added to the IP

Add more antibody to the IP

Antibody did not function in an IP

Verify that the antibody is qualified for ChIP or IP applications and has been handled and stored properly

Agarose ChIP Kit／Magnetic ChIP Kit を用いた ChIP アッセイで、ポジティブコントロールとネガティブコントロールの qPCR シグナル（サイクルスレッシュホールド）が非常に近いか違いがみられません。どういった理由が考えられますか？

Verify that your specific antibody (if not using the kit-provided RNA polymerase II antibody) is validated for IP. Ideally, a ChIP validated antibody is the best, but an antibody for IP has a good chance of working in ChIP.
Ensure that your chromatin is properly digested (see Appendix A in the manual). Too much digestion as well as too little digestion will affect the success of the ChIP reaction.
Ensure that all the chromatin has been released from the nuclei. When following the Magnetic ChIP kit instructions, MNase digestion of 4x10⁶ cells followed by sonication to lyse the nuclei, yields about 20-50 µg for the IP. This same sequence can be used with the Agarose ChIP Kit as well. It is recommended that you start with 2–4 x 10⁶ cells per ChIP reaction. Once a successful ChIP has been run at this cell number, it is possible to decrease the cell amount empirically. We have seen good results using as little at 10,000 cells, but this entirely depends on the cell line, target, and antibody.
Ensure that enough DNA was used for qPCR. Typically, 30-80 ng of DNA is a good range.

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