ChIp Troubleshooting Guide

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.

ELISA Troubleshooting Matrix Effect

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.

ELISA Troubleshooting Low Signal

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

IHC Non-specific Staining

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

IHC Troubleshooting: Overstaining

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

IHC Troubleshooting: Weak Staining

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

IHC Troubleshooting: High Background

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)

Western Blot Troubleshooting Distorted Bands

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

Western Blot Troubleshooting High Background

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

Western Blot Troubleshooting Background Background Blotchy, Flecked, Or Dirty

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