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FAQ

1. Low / No Signal

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Not enough cells/chromatin	Add enough chromatin for each IP experiment. We suggest using at least 25 μg of chromatin for each IP.
   ►	Incorrect Protein A/G/L used	Make sure that the Protein A/G/L beads are capable of binding to the antibody subclass being used.
   ►	Cross-linking process too long	Over Cross-linking with formaldehyde might mask the antibody binding sites and reduce antibody binding ability. It is advisable to optimize the cross-linking steps by using different concentration of formaldehyde or reducing cross-linking time.
   ►	Not enough antibody	Titre antibody amount used for each IP to determine the optimal condition. Up to 10ug of antibody can be used for each IP experiment.
   ►	Washes too stringent	Reduce the number of washes. Reduce salt concentration in the wash buffer.
   ►	Antibody not capable of immunoprecipitation	Try a different antibody. Try polyclonal antibody if monoclonal antibody does not work well.
   ►	The chromatin size might be too small	Make sure that the shearing condition is not too harsh which might results in fragments of DNA smaller than what the primers are able to amplify.
   ►	Incomplete elution from the Protein A/G/L beads	Incubate beads in elution buffer at 65°C with frequent mixing.

2. Positive signal seen in no template control

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	PCR reagent might be contaminated	Prepare new solutions from stock.

3. High Background observed in negative control

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Inadequate washing	Use a more stringent washing buffer. Try to  use a high salt washing buffer or increase the number of washes.
   ►	Non specific binding to Protein A,G or L	Include a pre-clear step by incubating lysate with Protein A/G/L agarose beads.
   ►	Too much DNA template added to the PCR reaction, or too many cycles of amplification	Add less DNA template or reduce the number of cycles of amplification.  Alternatively, real-time PCR can be used for the detection of ChIPed DNA products.
   ►	Buffers may be contaminated	Use freshly prepared lysis or wash buffers.

1. No binding

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Insufficient antibody	Check the recommended amount of antibody as indicated in the datasheet.  Titrate and optimize the optimal antibody amount used per IP experiment.
   ►	Washes too stringent	Reduce the number of washes. Reduce salt concentration in the wash buffer.
   ►	Incorrect Protein A/G/L used	Make sure that the Protein A/G/L beads are capable of binding to the antibody subclass being used.
   ►	Target protein not present in the sample used	Make sure that the target protein is expressed at a relatively high level in the sample used by including an Input sample in the WB.
   ►	Incorrect Lysis buffer used	Make sure that the lysis buffer used is not over-denaturing and destroy the native conformation of the target proteins.
   ►	Antibody not capable of immunoprecipitation	Try a different antibody. Try polyclonal antibody if monoclonal antibody does not work well.

2. Interference from heavy or light chain

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Secondary antibody rcognizes heavy / light chain denatured from primary  antibody	Use secondary antibodies which only recognizes native form of IgG for immunoblotting.

3. High Background

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Inadequate washing	Use a more stringent washing buffer. Try to  use a high salt washing buffer or add 0.2% SDS or 1% Tween20 to washing buffer. Increase the number of washes.
   ►	Concentration of antibodies too high	Check the recommended amount of antibody as indicated in the datasheet.  Titrate and optimize the optimal antibody amount used per IP experiment.
   ►	Non specific binding to Protein A,G or L	Pre-block beads with BSA. Incubate beads with 2%BSA in PBS for 1 hour wash in PBS before use.
   ►	Non specific binding to agarose beads	Include a pre-clear step by incubating lysate with Protein A/G/L agarose beads.
   ►	Antibody not specific enough	Use affinity purified and pre-absored antibody for IP experiments.
   ►	Sample degradation	Add adequate protease inhibitors and phosphatase inhibitors throughout sample preparation and Ip steps.

1. Molecular Weight Different from Predicted

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Protein post-translationally modified or alternatively spliced	Check if the protein of interest is post-translationally modified or alternatively spliced and produce other isoforms.
   ►	Incomplete protein denaturation	Freshly add DTT or β-Mercaptoethanol to sample buffer, or adequaltely boil samples to ensure complete bond breakage between peptides.
   ►	Membrane protein issue	If a membrane protein is to be detected, try low temperature (~65°C) or avoid boiling which might cause aggregation of membrane proteins.

2. Multiple bands

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Protein post-translationally modified or alternatively spliced	Mix gel completely before pouring. If SDS-PAGE should be run the day after preparation, make sure that the gel is prepared at RT and store in moist chamber at 4°C.
   ►	Protein degradation	Keep the voltage and temperature low while running SDS-PAGE. If necessary, run gel in the cold room. Remove bubbles trapped at the bottom of gel to ensure even electrophoresis.
   ►	Protein Multimerization	Make sure that the salt concentration of lysis buffer is kept between 0.15M to 0.5M.
   ►	Antibody concentration too high	Make sure that total amount of protein loaded into each well is between 20-50μg.
   ►	Antibody issue	Optimize the dilution factor of primary and secondary antibody.
   ►	Interference from secondary antibody	While performing Immunoprecippitaiotn (IP) experiment, make sure that the secondary antibody used to detect the protein of interest is derived from a species different from that of antibody used to pull down protein. Alternatively, use a secondary antibody that recognize only the native form of IgG to detect IP protein.

3. Black Dots

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Reagent contaminated	Prepare all reagents freshly.
   ►	Blocking agent insufficiently dissolved	Make sure that the blocking agent such as milk or BSA is completely dissolved before use. Alternatively, filter the blocking solution with 0.45μm filter before use.

4. Band Artifacts (White bands, smile effect, streaks)

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Poor gel preparation	Mix gel completely before pouring. If SDS-PAGE should be run the day after preparation, make sure that the gel is prepared at RT and store in moist chamber at 4°C.
   ►	Voltage, temperature too high, field effect	Keep the voltage and temperature low while running SDS-PAGE. If necessary, run gel in the cold room. Remove bubbles trapped at the bottom of gel to ensure even electrophoresis.
   ►	High salt concentration in samples	Make sure that the salt concentration of lysis buffer is kept between 0.15M to 0.5M.
   ►	Protein overloaded	Make sure that total amount of protein loaded into each well is between 20-50μg.
   ►	Antibody concentration too high	Optimize the dilution factor of primary and secondary antibody.

5. Smeary Staining

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Poor gel preparation	Mix gel completely before pouring. If SDS-PAGE should be run the day after preparation, make sure that the gel is prepared at RT and stored in moist chamber at 4°C.
   ►	Protein overloaded	Make sure that total amount of protein loaded into each well is between 20-50μg.
   ►	High membrane protein concentration in samples	If membrane fraction is used, make sure that sample is sufficiently diluted before loading into SDS-PAGE.

1. Poor standard Curve

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Improper standard dilution	Use appropriate diluent as blank. Make sure that the dilution is performed as according to protocol
   ►	Standard improperly reconstituted	Briefly spin standard vial before opening. Make sure that there is no undissolved material after reconstituting
   ►	Standard degraded	Store standards as according to protocol
   ►	Curve doesn’t fit the scale	Try plotting log-log or 5 parameter logistic curve fit
   ►	Pipetting error	Calibrate pipettes to make sure that the correct volume is dispensed
   ►	Incomplete washing	Increase washing cycles

2. Variation among replicates

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Improper washing	Make sure that the washing is done as according to protocol
   ►	Poor mixing of samples	Mix samples gently and evenly
   ►	Dirty plate	Make sure that the bottom of plate is clean
   ►	Reagents too old	Make sure that the reagents are not expired. Use freshly prepared reagents
   ►	Bubbles in wells	Make sure that there is no bubble in wells before reading
   ►	Inconsistent pipetting	Calibrate pipettes to make sure that the correct volume is dispensed
   ►	Edge effects	Make sure that the plate and reagents are equilibrated to room temperature before starting assay

3. High background

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Too much antibodies was used	Reduce the concentration of primary or secondary antibodies
   ►	Antibodies bind nonspecifically	Use blocking buffer or choose another affinity-purified antibody
   ►	Too much substrate reagent used	Use substrate with higher dilution
   ►	Insufficient washing	Increase washing cycles
   ►	Wrong concentration of blocking reagent	Check the recommended concentration of blocking buffer
   ►	Reaction not stopped	Stop reactions with STOP buffer before reading
   ►	Plate left too long before reading	Take measurements shortly after addition of substrate and STOP buffer
   ►	Insufficient Tween in wash buffer	Use PBS+0.05% Tween as wash buffer
   ►	Incubation temperature too high	Optimize incubation temperature for each experiment
   ►	Plate stacking during incubation lead to uneven temperature throughout the plate	Avoid stacking plates together during incubation
   ►	Pipetting error	Calibrate pipettes to make sure that the correct volume is dispensed
   ►	Reagents not mixed properly	Make sure that all reagents are mixed properly and equilibrated to room temperature before assay
   ►	Salt concentration of incubation and wash buffer	Increase salt concentration to reduce nonspecific interaction
   ►	Substrate incubation carried out in light	Perform substrate incubation in dark
   ►	Dirty plate	Make sure that the bottom of plate is clean

4. Weak signal

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Insufficient coating	Use more antigens or antibodies for coating
   ►	Substrate reagents have expired or prepared at a wrong pH	Use fresh substrate reagents

5. No signal

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Assay set up incorrectly	Make sure that the instructions in the protocol is followed carefully
   ►	Incorrect secondary antibody used	Check if the correct secondary antibody is used
   ►	Insufficient antibodies used	Increase concentration of primary or secondary antibody
   ►	Substrate reagents not fresh	Use fresh substrate reagents
   ►	Wrong settings of plate reader	Check the settings (wavelength, filters, gain etc) of plate reader
   ►	Insufficient incubation	Follow the incubation time as indicated in the protocol booklet
   ►	Sample concentration falls below detection limits of kit	Decrease dilution factor or concentrate samples
   ►	Plate washing too vigorous	Check the setting of plate washer. Pipette wash buffer into wells gently
   ►	Wells dried out	Cover plate with adhesive film or incubate in humidified chamber throughout experiment
   ►	Enzyme inhibitor present in buffers or reagents	Inhibitors such as Sodium Azide can affect enzyme and assay performance. Ensure that there is no enzyme inhibitor in any buffers

1. High background

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Insufficient blocking	Select appropriate serum as blocking buffer. Blocking for 1 hour at room temperature.
   ►	Interference from endogenous enzymes	Perform H2O2 or Levamisole quenching.
   ►	Non-specific binding from primary antibodies	Dilute primary or secondary antibody. Choose another IHC-validated primary antibodies.
   ►	Inadequate washing	Increase washing cycles/time. Increase salt/detergent concentration for stronger washes.
   ►	Non-specific binding from secondary antibodies	Perform secondary antibody incubation only. Use pre-adsorbed secondary antibody.
   ►	Non-specific binding from chromogen	Perform chromogen incubation only. Use other chromogen if necessary.
   ►	Interference from secondary antibody in multicolor staining	Make sure that the fluorochrome does not overlap with one another.
   ►	Autofluorescence issue	Make sure that there is no endogenous background caused by tissue itself. Check under fluorescence microscope prior to staining to identify autofluorescence.

2. Problem with morphology

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Antigen retrieval too harsh	Optimize retrieval steps to give the best morphology.
   ►	Tissue sections peeled off slide	Dry samples for 2-4 hours at 60°C. Tissue with high lipid content (eg breast tissues) should be dried for longer time.
   ►	Sectioning issue	Cut thinner slides for better resolution: 3-5 μm. Use a new/sharper blade.
   ►	Autolysis has occurred	Fix samples as soon as possible. Choose other fixatives to accelerate penetration. Fixative perfusion might be necessary for larger tissues.

3. Low/No signal

   Check the possible causes and find out the solution!

   	Possible Causes	What You Can Do?
   ►	Over fixation	Reduce fixation time. Perform antien retrieval to unmask epitopes.
   ►	Insufficient fixation	Increase fixation time or try other fixative.
   ►	Fixation process delayed	Fix immediately as tissue is extracted.
   ►	Permeabilization issue	For nuclear/cytoplasmic proteins, add permeabilization agent (eg Triton X-100, Saponin) in blocking and antibody incubation buffer.  For membrane/tight junction proteins, avoid permeabilization agent.
   ►	Primary antibodies not suitable for IHC	Choose an IHC-validated primary antibodies.
   ►	Wrong secondary antibody used	Make sure that primary and secondary antibodies match one another.
   ►	Low expression of protein in tissue samples	Use signal amplification methods (eg: HRP Polymer ARG80982, ARG80967, ARG80966)
   ►	Insufficient deparafinization	Make sure that parafin is removed completely before staining.