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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, 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.

 

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.