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DNA EMSA
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.
|
The shift may be caused by non-specific binding or a DNA binding protein different than the one being tested.
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.
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.
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
|
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.
Use a non-specific competitor DNA such as poly(dI.dC).
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
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.
|
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
|
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.
Use a nonspecific competitor RNA such as tRNA or heparin.
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
- Work in a clean nuclease-free environment.
- Ensure that all plastics are nuclease-free.
- After in vitro transcription, check RNA integrity by gel electrophoresis.
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.
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.
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
|
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 |
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
|
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
|
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
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.
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.
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)
|
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).
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.
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
|
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.
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)
|
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 |
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.
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
|
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
|
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.
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
|
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
|
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.
- 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.
For Research Use Only. Not for use in diagnostic procedures.