ChIP sequencing (ChIP-Seq) is a powerful method for identifying genome-wide DNA binding sites for transcription factors and other proteins. Use this guide to help troubleshoot common issues that arise during ChiP procedures:
High Background
- Pre-clear lysate sample. Clear lysate with protein A/G affinity beads to remove proteins that nonspecifically bind and increae background signal.
- Use fresh buffers Contaminated buffers can cause increased background. Prepare fresh lysis and wash buffers to eliminate this source of error.
- Use high-quality protein A/G beads. Low-quality protein A/G beads can give high background signal. Use Boster’s quality-guaranteed protien A/G beads to ensure good results.
Low Signal
- Reduce sonication. Excessive sonication can result in low fragment sizes that produce poor results. Optimize sonication time to yeild fragments between 200-1000 bp.
- Improve cell lysis. Insufficent lysis will result in low signal. Use Boster’s high-quality lysis buffers to ensure good results
- Reduce cross-linking intensity. Excessive cross-linking by formaldehyde fixation can mask epitopes and reduce signal intensity. Reduce fixation time and quench with glycine to increase signal.
- Use more starting material. Too little starting material will yeild poor results. We recommend using 25 ug of chromatin per immunoprecipitation.
- Use more antibody. Increase amount of antibody used to boost signal. We recommend between 1-10ug of antibody to maximize results.
- Reduce salt concentration of wash buffers. Wash buffers with excessive osmolarity can reduce antibody binding activity. We recommend using buffers with no more than 500 mM salt.
Low Resolution and High Background
- Reduce DNA fragment size. Optimize sonication to acheive fragment length of 200-1000 bp.
Matrix Effect occurs when the target antigen interacts with matrix components in plasma or serum samples. These matrix components can be endogenous biological components such as phospholipids, carbohydrates, and metabolites. Matrix components can reduce the binding of the antibody to the target protein, or non-specifically bind the antibody, generating weak or noisy results.
Here are some tips to reduce matrix effect:
- Centrifuge the sample. Centrifugation can separate matrix components from soluble antigens, reducing the concentration of matrix components and their effect on results.
- Increase dilution. Increasing the dilution factor 2-5 fold will reduce matrix component binding and mitigate the matrix effect. Note: when diluting the samples remember to use the same diluents as used for the standard curve.
Weak or no color development in an ELISA assay can indicate that the target protein is present in minute quantities in the sample, if at all. It can also mean that there is something wrong with the assay or the reagents that prevents efficient detection. If your control reactions indicate that an error is causing your poor results, use this troubleshooting guide to diagnose and resolve your ELISA weak signal.
| Problem | Cause | Solution |
|---|---|---|
| Antibody/epitope reaction problems | Capture antibody failed to absorb to plate | Coat the plate for longer |
| Use more concentrated coating components | ||
| Use Boster pre-coated ELISA Kits | ||
| Epitope recognition impeded by absorption to the plate | Conjugate target protein to carrier peptide before coating to plate | |
| Primary antibody concentration too low | Increase primary antibody concentration | |
| Incubate for longer | ||
| Loss of binding activity due to improper storage | Store antibodies ant -20C or below | |
| Avoid repeated freeze-thaw cycles | ||
| Insufficient reporter enzyme activity | Enzyme inhibitor present | Avoid sodium azide in HRP reactions |
| Avoid phosphate in AP reactions | ||
| Detection reagent old, contaminated, or wrong PH | Use fresh substrate at the correct pH | |
| Detection substrate too dilute | Increase concentration of detection substrate | |
| Incorrect incubation temperature | Optimize incubation temperature | Make sure all reagents are at room temperature before beginning |
| Plate error | Excessive washing | Calibrate automatic washer to the correct pressure |
| Wash gently with a manual pipette | ||
| Wells dried out | Cover the plate with sealing film during incubations | |
| Well bottoms scratched by pipette tips | Use caution when dispensing reagents |
Immunohistochemistry (IHC) is one of the many methods that researchers use to visually detect specific antigens in a sample. A variety of issues can arise during the staining step of IHC, such as non-specific staining. Non-specific staining occurs when the primary antibodies bind to proteins other than the target protein, resulting in data unusable for meaningful interpritation.
Here are some tips to reduce non-specific binding when using IHC:
| Problem | Cause | Solution |
|---|---|---|
| Improper Sample Preparation | Inadequite Deparaffinization of the tissue section | Increase deparaffinization time |
| Use fresh dimethylbenzine | ||
| Inadequite quenching of endogenous peroxidases or biotins | Use H2O2 to quench endogenous peroxidase activity | |
| Block endogenous biotin with excess free avadin | ||
| Insufficient blocking | Increase blocking time | |
| Section dried out | Avoid allowing your tissue section to dry out | |
| Insufficient washing | Increase washing time and number of washes | |
| Antibody problems | Antibody contaminated | Affinity purify your antibodies |
| Use Boster high-quality antibodies | ||
| Excessive primary antibody concentration | Reduce antibody concentration |
Overstaining occurs due to excessive development of signal in the sample. This causes the sample to become saturated, reducing contrast. Overstained samples can appear blurry, diffuse, or monochromatic. Overstaining can prevent accurate visualization of tissue structures and inhibit useful detection of protein localization.
Here are some tips to reduce overstaining when using IHC:
| Causes | Solutions |
|---|---|
| Primary antibody too concentrated | Dilute primary antibody solution |
| Perform a titration to determine optimal antibody dilution | |
| Excessive primary antibody binding | Reduce the length of the incubation step |
| Incubate in a cold room at 4C | |
| Detection substrate incubation time too long | Allow less time for signal development after adding the detection substrate |
| Insufficient washing | Increase number and time of washes |
Good results in IHC experiments depend on strong, specific staining of the target antigens. A good result can only be achieved when a sufficient quantity of primary antibody penetrates the sample and binds its target with high specificity, and enough secondary antibody with active enzymatic or fluorescent conjugate binds the primary antibody. When an IHC experiment results in faint or weak signal, it often has to be repeated, costing valuable time, money, and resources. Use this troubleshooting guide to identify and resolve the source of your weak signal:
| Problem | Cause | Solution |
|---|---|---|
| Low enzyme activity | Enzyme/substrate reaction impeded | Deionized water can sometimes contain peroxidase inhibitors. Use Boster antibody diluent buffer to ensure enzymatic activity |
| Optimize substrate pH to increase reaction intensity | ||
| Buffer incompatible with enzyme | Do not use phosphate buffer with AP system | |
| Do not use sodium azide with HRP system | ||
| Insufficient anitbody activity | Loss of antibody potency due to time or improper storage | Run a positive control to verify antibody binding activity |
| Store antibodies according to manufacturer instructions | ||
| Inadequate antibody penetration | Use a permeabilizing agent in the blocking and antibody diluent buffers | |
| Antibody concentration too low | Use a higher antibody concentration or incubate for longer | |
| Antibody cannot detect target protein in native conformation | Check the datasheet to make sure your antibody has been validated for IHC | |
| Check that the antibody is applicable to your IHC sample (paraffin vs. frozen sections) | ||
| Perform western blot on both native and denatured forms to verify antibody binding | ||
| Problems with tissue section | Insufficient deparaffinization | Increase deparaffinization time |
| Use fresh dimethylbenzene | ||
| Inadequate antigen retrieval | Reduce the length of fixation step | |
| Use a different antigen retrieval method | ||
| Perform antigen retrieval for longer | ||
| Tissue section has dried out | Make sure the tissue is covered in liquid at all times | |
| Loss of signal over time | Prepare slides using fresh tissue sections | |
| Store slides at 4C | ||
| Do not bake slides |
You can typically expect some amount of background staining during IHC. However, once the level of background staining becomes high enough to obscure important features and structures of the tissue, steps must be taken to reduce it. Background staining can be caused by inappropriate antibody binding or by mistakes during the preparation of the tissue slide. Use this guide to resolve your high background staining:
| Problem | Cause | Solution |
|---|---|---|
| Antibody binding incorrectly | Excessive primary antibody concentration | Titrate antibody to determine optimal concentration |
| Secondary antibody non-specific binding | Run a negative control without primary antibody to assess secondary antibody binding | |
| Use a secondary antibody pre-adsorbed against the immunoglobulin of the sample species | ||
| Block your sample with serum from same host as secondary antibody | ||
| Antibody cross-reactivity | Use Boster primary antibodies guaranteed specific to only their listed targets | |
| Improper preparation of tissue section | Insufficient blocking | Increase blocking incubation time |
| Use a different blocking reagent | ||
| Insufficient quenching of endogenous peroxidase or phosphatase | Quench endogenous peroxidase with 3%H2O2 solution | |
| Quench endogenous alkaline phosphatase with 2mM levamisole | ||
| Insufficient biotin or lectin blocking | Block endogenous biotin with=streptavidin solution | |
| Block endogenous lectins with alpha-methyl mannoside buffer | ||
| Incomplete deparaffinization | Increase deparafinization time and use fresh dimethylbenzene | |
| Tissue section too thick for complete reagent penetration | Prepare thinner sections | |
| Improper incubation parameters | Incubation temperature too high | Incubate at 4C |
| Too much substrate was used | Reduce substrate concentration | |
| Reduce substrate incubation time | ||
| Choose a substrate with higher signal:noise ratio (e.g. metal-enhnaced DAB) |
Distorted bands can make it very hard to interpret your results. Common distortions include smile shaped bands with the edges trailing upward, diffuse bands that are broad or blurry, and streaked bands that trail off in several directions. Make sure your next blot has even, crisp bands by following these tips:
| Problem | Cause | Solution |
|---|---|---|
| Curved, “smiling” bands | Electrophoresis voltage too high | Reduce voltage; run the gel slower for more consistent results |
| Overheated gel | Reduce voltage or run the gel on ice or in a cold room (-4C) | |
| Streaked or diffuse bands | Incomplete contact between gel and membrane during transfer | Use thicker filter paper in sandwich |
| Squeeze air bubbles and excess buffer from between membrane and gel | ||
| Slippage of membrane during transfer | Avoid agitating or moving the gel or membrane during transfer | |
| Blurry bands | Electrophoresis voltage too high | Run the gel for longer at a lower voltage |
| Impropher loading buffer composition | Mix new loading buffer, or use Boster formulated loading buffer | |
| Air bubbles trapped between gel and membrane during transfer | Carefully remove bubbles by squeezing them out with a sterile glass rod | |
| Ghost bands | Overexposure during visualization | Decrease the exposure time |
| Loading sample too concentrated | Reduce the amount of sample loaded. See Boster’s Western blot troubleshooting guide for load optimization tips | |
| Antibody concentration too high | Dilute your antibody solutions using Boster’s antibody diluent medium | |
| Blot was moved during transfer | Avoid agitating or moving the gel or membrane during transfer | |
| Uneven, crooked bands | Poor gel polymerization | Check gel concentration and ensure complete dissolution of SDS before casting gels |
| Varying salt concentration between wells | Make sure the salt concentration in different samples is similar |
Typically, polyacrylamide gel electrophoresis separates proteins by molecular weight, with large proteins migrating slower than small proteins. This process is can be affected by several factors: protein degradation can cause proteins to appear shorter than the expected length, glycosylation can cause proteins to appear larger than predicted, and nonspecific antibody binding can cause multiple bands to appear on a blot where only one is expected. Use these tips to identify and resolve the source of your unexpected band sizes.
| Problem | Cause | Solution |
|---|---|---|
| Blocking issues | Too little membrane blocking | Increase the concentration of blocking solution |
| Increase the temperature at which the blocking is performed | ||
| Increase the time spent blocking | ||
| Blocking proteins reacting with detection system | Milk contains biotin; do not use when detecting with avadin-biotin system | |
| Try one of Boster’s other blocking reagents | ||
| Contaminated blocking solution | Never re-use blocking solutions | |
| Use pure protein as a blocking agent | ||
| Incubation issues | Excessive substrate incubation | Reduce length of substrate incubation |
| Contaminated incubation equipment | Use disposable incubation trays | |
| Thoroughly wash reusable incubation trays between incubations | ||
| Insufficient membrane washes between incubations | Increase washing stringency | |
| Incubation temperature too high | Incubate for longer at a lower temperature | |
| Detection issues | Membrane overexposed | Check exposure parameters and try a shorter exposure time |
| Insufficient antibody binding activity | Use fresh aliqot of antibody stored at -20C | |
| Avoid thawing and re-freezing antibodies | ||
| Store antibodies at -80C for long-term stability | ||
| Excessive antibody concentration | Reduce antibody concentration | |
| Use a dot-blot test to optimize antibody concentration |
The background of a western blot does not always appear clean and flawless. Blotches, streaks, and spots are all common artifacts that can make it hard to interpret and publish your results. These artifacts are most commonly the result of uneven coating of buffer or antibody, the membrane drying out, or aggregates forming in the antibody or blocking buffer. Follow these tips to identify and solve the cause of your imperfect western blot background:
| Problem | Cause | Solution |
|---|---|---|
| Blotched background | Uneven antibody distribution | Agitate during incubation to coat the membrane evenly in incubation buffer |
| Membrane dried out unevenly | Make sure membrane is thoroughly wetted before starting the protocol | |
| Ensure the membrane does not dry out during any step | ||
| Uneven wash/incubation buffer coverage | Increase volume of wash and incubation buffers | |
| Do not stack membranes during incubation | ||
| Flecked background | Secondary antibody aggregation | Increase secondary antibody dilution to prevent aggregation |
| Spin down or filter out antibody aggregates | ||
| Clumps of blocking buffer binding secondary antibody | Use fresh blocking buffer | |
| Increase Tween 20 concentration of blocking buffer | ||
| Filter blocking buffer before use | ||
| Use a different blocking reagent, such as albumin, BSA, or casein | ||
| Wash membrane with wash buffer before antibody incubation | ||
| Buffer contamination | Mix new buffers | |
| Filter buffers before use | ||
| White spots with no protein transfer | Air bubbles trapped between gel and membrane during transfer | Carefully squeeze out bubbles from between membrane and gel using a sterile glass rod |
| Use enough buffer to saturate the membrane |
Typically, polyacrylamide gel electrophoresis separates proteins by molecular weight, with large proteins migrating slower than small proteins. This process is can be affected by several factors: protein degradation can cause proteins to appear shorter than the expected length, glycosylation can cause proteins to appear larger than predicted, and nonspecific antibody binding can cause multiple bands to appear on a blot where only one is expected. Use these tips to identify and resolve the source of your unexpected band sizes.
| Problem | Cause | Solution |
|---|---|---|
| Bands have higher MW than expected | Proteins are glycosylated or bear other post-translational modifications | Review literature and identify modified forms of your target protein |
| Strip post-translational modifications with enzymatic treatment | ||
| Bands have much higher MW than expected | Protein aggregation | Decrease protein concentration |
| Prepare new sample with fresh loading buffer | ||
| Incomplete denaturation or residual disulfide bonding | Denature the protein with urea | |
| Use stronger reducing agents | ||
| Use fresh 2-mercaptoethanol or DDT to strip disulfide bonds | ||
| Bands have lower MW than expected | Protein sample has been digested or degraded | Use fresh sample from frozen stock |
| Use a lysis buffer with proteinase inhibitors | ||
| Primary antibody is detecting splice variants | Review literature to identify splice variants of your protein | |
| Try a different primary antibody | ||
| Primary antibody binding a similar epitope on a different protein | Run a negative control to detect other proteins that react with your antibody | |
| Multiple bands | Primary or secondary antibody contaminated with nonspecific IgG | Use Boster primary antibodies guaranteed free of nonspecific IgG |
| Nonspecific binding of primary antibody | Increase antibody dilution | |
| Affinity purify primary antibody to select for only desired binding activity | ||
| Use Boster primary antibodies guaranteed to only bind their indicated targets | ||
| Nonspecific binding of secondary antibody | Reduce antibody concentration | |
| Run a negative control with just the secondary antibody to detect nonspecific binding | ||
| Insufficient blocking | Use higher concentration blocking buffer | |
| Block for longer | ||
| Add tween 20 to blocking buffer | ||
| Ionic interactions | Increase stringency of washing step | |
| Increase salt concentration of incubation buffers | ||
| Include stronger detergents in the washes |
A weak western blot signal is characterized by faint or indistinct bands. While the bands may be barely visible at their predicted sizes, weak signal can often require repeating the experiment. Common sources of this error occur during protein transfer and detection. Use these tips to identify the source of the error and get better results:
| Transfer issues | Insufficient sample concentration | Increase the amount of starting material |
| Concentrate your sample using immunoprecipitation or similar procedure | ||
| Transfer too vigorous | Reduce transfer time or voltage to prevent small proteins transferring completely through membrane | |
| Use a secondary membrane to capture proteins transferred through the primary membrane | ||
| Use a membrane with smaller pore size | ||
| Inadequate transfer | Increase transfer time or voltage | |
| Sandwich assembly oriented incorrectly | Make sure the sandwich assembly is oriented correctly relative to the electric field | |
| Check the polarity of the electric field | ||
| Incorrect transfer buffer PH | Adjust transfer buffer PH to be 2 points lower than the pI of protein sample to optimize charge:mass ratio | |
| Detection issues | Insufficient antibody concentration | Use a dot blot assay to optimize protein concentration |
| Insufficient antibody binding affinity | Reduce washing stringency | |
| Increase antibody concentration | ||
| Use Boster high affinity primary antibodies | ||
| Insufficient sample loading | Use more starting material | |
| Concentrate sample prior to loading | ||
| Antigens masked by dry milk blocking solution | Nonfat dry milk can sometimes mask antigens. Try using a different blocking reagent |