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Protocol Tips and Tricks

 

Western Blotting / Immunoblotting (WB / IB)

General Tips to concider when performing a western blot:

  • Increasing/decreasing primary antibody concentration can help amplify/reduce signal, respectively.
  • Increasing/decreasing enzyme substrate incubation time can help amplify/reduce signal, respectively.
  • Always run a positive control to ensure antibody activity (purified protein, transfected lysate, etc.).
  • More/longer washing steps can help reduce both specific and nonspecific antibody staining.
  • Efficient protein of interest transfer is dependent on 1) protein size, 2) transfer power settings, and 3) transfer time.

Aviva's Western Blot Conditions:

~25 ug/lane = Amount Cell/Tissue Lysate Protein Used Per Lane

Typical antibody concentration range = 0.2 - 5.0 ug/mL, or begin with a 1:1000 dilution of antibody reconstituted at 1 mg/mL.  The optimal antibody concentration must be determined empirically.

Why is the actual western blot band size different from the predicted?

Western blotting is a technique that separates proteins based on size. In general, the smaller the protein the faster it migrates through the gel. However, migration is also affected by other factors, so the actual band size observed may differ from that predicted. Common factors include:

Post-Translational Modification: e.g., Phosphorylation, glycosylation etc., which increases the size of the protein.

Post-Translational Cleavage: e.g., Many proteins are synthesized as pro-proteins and then cleaved to give the active form, e.g. pro-caspases.

Splice Variants: Alternative splicing may create different sized proteins produced from the same gene. These splice variants can result in the production of different sized protein products. Expression of splice variants is highly variable depending on the specific tissue and experimental conditions being used. Several web sites can be accessed to determine splice variants for specific targets.

Isoforms: Many proteins express multiple isoforms that are different sizes. Expression of different isoforms for the same target protein is highly variable depending on the specific tissue and experimental conditions being used. Several web sites can be accessed to determine isoforms for specific targets.

Relative Charge: The composition of amino acids (charged vs non-charged).

Multimers: e.g., Dimerization of a protein. This is usually prevented in reducing conditions, although interactions can result in the appearance of higher bands.

Why do I see a weak signal or no signal at all on my western blot?

Antibody Titration: A titration experiment should be performed with multiple antibody concentrations to determine the appropriate antibody concentrations for optimal signal/noise ratios and for obtaining the best signal. In general this titration should range from 0.2 to 5.0 ug/ml.

Tissue/Cell Specificity: Target protein expression is dependent on the cell or tissue being examined. A literature search should be conducted to ensure that the system that you are using is appropriate for detecting the target protein in question. Several web sites can be accessed to determine general mRNA expression patterns for specific targets which may serve as a general guideline for target protein expression, although mRNA expression does not always correlate directly to protein expression (http://www.genecards.org/index.shtml).

Reconstitution: Care should be taken in re-suspending lyophilized antibodies in order to ensure that the antibody is appropriately resolublized. Although the lyophilized antibody pellet is often at the bottom of the tube, in some cases the pellet may have localized on the tube wall. Consequently, the resolublization should cover the entire surface of the tube to ensure that the antibody is completely resolublized. The tube should then be briefly centrifuged to ensure that the entire re-solubilized pellet is collected at the bottom of the tube

Positive Control: The addition of a positive control lane to your western blot assay is the optimal means of assessing whether the antibody is functioning appropriately and the experimental conditions are appropriate.

All of Aviva Systems Biology antibodies have been tested and validated with a specific cell/tissue lysate that is indicated on the antibody specification data sheet.

Many of these cell lysates are available for purchase from Aviva Systems Biology and can be used in your experiment to ensure that the appropriate signal is achieved in your experiment.

Alternatively, a positive control based on the literature or internal experimental results can be used to assess whether the antibody is functioning appropriately.

Will this antibody react with the same target in a different species?

Primary Species Reactivity: Primary species reactivity for all Aviva Systems Biology antibodies is determined by western blot analysis.This is primarily done using a cell line or tissue derived from the primary species. The primary species reactivity as well as the validating tissue used are listed on the data sheets for each antibody that can be found on the ASB web site.

Secondary Species Reactivity: All secondary species reactivities were determined by computational sequence homology analysis, comparing the homology between the immunizing peptide sequence and the NCBI database for each specific species.

Why are there extra bands on my western blot?

If non-specific binding of primary antibody, you can:

  • Reduce primary antibody concentration
  • Reduce the amount of total protein loaded on gel
  • Use monospecific antibody, or affinity (antigen) purified antibodies

If non-specific binding of secondary antibody, you can:

  • Run a secondary antibody only control (w/o primary) and develop blot normally.  If background bands appear, use different secondary
  • Use monospecific antibody, or affinity (antigen) purified antibodies

If non-specific binding by primary or secondary antibodies, you can:

  • Add 0.1 - 0.5%  Tween20 to antibody dilution/wash buffers
  • Adjust % of milk in various buffers.  Start with 2% non-fat dry milk as Blocking buffer/antibody dilution buffer.  Optimize by increasing or decreasing % of milk
  • Increase number/time of washes
  • Increase NaCl concentration in blocking/antibody dilution/wash buffers to 0.15 - 0.5 M

If you detect what appears to be aggregation of your analyte, try:

  • Increase the amount of DTT (or BME) to ensure complete reduction of all disulfide bonds (20-100mM DTT).  Heat in 95-100C water for 5-10 min before loading onto gel
  • Quickly spin your sample before preparing for gel loading

To avoid "splotchy" or uneven blots when performing a western blot:

  • Extend the incubation with your blocking buffer
  • Increase the number/time of your washes (this is particularly important for tissue homogenate samples)
  • Ensure the milk powder is completely dissolved before adding to blot
  • Spin the tube containing the antibody solution before transferring for use, in case of protein aggregation.

To avoid patchy, uneven spots appearing all over blot, try:

  • Check all buffers used for bacterial contamination
  • Make sure membrane is fully immersed during antibody and washing incubations
  • Tease out all air bubbles between membrane and gel before performing transfer
  • Ensure uniform access to whole blot by placing membrane on a rocker/shaker
  • Ensure all western blotting equipment is properly washed
  • Filter HRP conjugate to remove any possible aggregates
  • Reduce substrate exposure time

Do my transfer conditions change if my protein of interest if >180kD?

  • Transfer conditions/buffers need to be optimized for every lab
  • Using 20% MeOH
  • Adding 0.05% SDS
  • Transfer times and power settings; try 1A for 1hr.
  • Increasing lysate amounts will increase the number of copies to be transferred to your membrane

 

Western Blot Protocols:

Western Blot Protocol: See 'Protocols and Procedures'

Blocking Peptide Competition Protocol: See 'Protocols and Procedures'

Additional references:

  • See product datasheet or contact Aviva Systems Biology

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 Immunohistochemistry/Immunocytochemistry (IHC/ICC):

Aviva IHC antibody concentrations:

Recommended Antibody Concentration: 0.5 - 5.0 ug/ml (1:200 to 1:2000)

Heat Induced Epitope Retrieval (HIER):

  • Antigen Retrieval Buffers
  • Citrate Buffer (acidic) (10mM Citric Acid, 0.05% Tween 20, pH 6.0)
  • Tris-EDTA Buffer (basic) (10mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0)
  • Tris-Urea Buffer (basic) (100 mM Tris, 5% (w/v) urea, pH 9.5)

1. Preheat the Antigen Retrieval Buffer to 95°C. This can be done by heating the buffer in a coverglass staining jar which is placed in a waterbath at 95°C.
2. Using a small pair of broad-tipped forceps, place the coverslips carefully in the Antigen Retrieval Buffer in the coverglass staining jar, making note of which side of the coverslips the cells are on.
3. Heat the coverslips at 95°C for 2-40 minutes.  Optimal times should be determined by the researcher.
4. Remove the coverslips from the Antigen Retrieval Buffer and allow them to come to room temperature.
5. Immerse coverslips with the side containing the cells facing up, in PBS, in the 6-well tissue culture plates.
6. Rinse the cells 3 times in PBS. 7. Continue with blocking and primary antibody staining steps.

Proteolytic enzyme Induced Epitope Retrieval (PIER):

  • TE Buffer (50mM Tris Base, 1mM EDTA, 0.5% Triton X-100, pH 8.0)
  • 20X Proteinase K Stock Solution (8mg Proteinase K @ 30units/mg, 10mL TE Buffer, 10mL glycerol)
  • Working Proteinase K Solution (1mL 20X Proteinase K, 19mL TE Buffer)
  • Calcium Chloride Stock Solution (0.1g CaCl2, 10mL H2O)
  • 10X Trypsin Stock Solution (50mg Trypsin, 10mL H2O)
  • Working Trypsin Solution (1mL 10X Trypsin, 1mL CaCl2 solution, 8mL H2O, pH 7.8)

1. Deparaffinize sections in 2 changes of xylene, 5 minutes each.
2. Hydrate in 2 changes of 100% ethanol for 3 minutes each, 95% and 80% ethanol for 1 minute each.
3. Rinse in distilled water.
4. Cover sections with Proteinase K or Trypsin working solution and incubate 10-20 minutes at 37C in humidified chamber (optimal incubation time should be determined by researcher).
5. Allow sections to cool at room temperature for 10 minutes.
6. Rinse sections in PBS Tween 20 2 times for 2 min/each.
7. Continue with blocking and primary antibody staining steps.

Should I use frozen or paraffin sections?

  • The most common histological preparative technique is formalin-fixed, paraffin embedded tissue.  Some epitopes are more sensitive to fixation and embedding than others and can be masked (hidden) from the addition of affinity reagents.  Various antigen retrieval methods exist to unmask a given epitope.  These are not applicable to frozen sections.  The optimal method will have to determined on a case by case basis.  Crosslinking when fixating and embedding can prevent antigen degradation or physical relocation within the cell/tissue.  It also eliminates bacterial contamination.  Frozen sections can often lose morphological integrity, whereas paraffin embedded sections tend to retain it over multiple sections. Paraffin embedded sections will cause cell/tissue shrinkage that results in higher antigen density over a given section. Always be aware of the fact that your tissues/cells may contain various components that could interfere with your staining technique, such as, endogenous enzymes (alkaline phosphatase, peroxidases), endogenous biotin and Fc receptors.

How can I increase my signal?

  • The use of an enzyme or fluorophore conjugated secondary polyclonal antibody that has been selected to bind specifically and with high affinity to many different epitopes of your primary antibody can drastically amplify signal. Utilizing the extremely high affinity interaction of biotin:avidin/streptavidin in your affinity reagents (labeling your primary antibody with biotin then using a streptavidin-HRP conjugate) can allow an increase in signal. Beware of endogenous biotin expression in your samples. Try a different antigen retrieval method. Some epitopes are more sensitive than others to different fixations and antigen retrieval methods.

Should I use IHC or IF?

  • IHC is relatively light-insensitive and allows the visualization of tissue architecture. Autofluorescence can sometimes make IF studies impossible. IF allows for staining of the same subcellular structure with different fluorophores.

Which is more sensitive, HRP or AP?

  • NBT/BCIP substrate for AP is the most sensitive but, not widely used because the reaction is slow, does not allow adequate nuclear counterstain, the signal can diffuse and is not compatible with permanent mounting media. Development using DAB substrate for HRP is far more common due to speed of reaction, precise deposition and acceptable color contrast with nuclear stains.

Should I use detergent to permeabilize the cells?

  • Upon fixation, all intracellular trafficking of molecules is stopped. Drying and lipid solvent treatments (acetone, ethanol, etc.) create massive holes in the greater sub/cellular structure. This allows antibodies to cross membranes (extracellular, nuclear, etc.) in fixed cells. There is no detergent that causes an antigen that was previously hidden (masked) to be exposed (unmasked). Low levels of detergents such as Tween 20 in the washing solutions reduce the surface tension and allow the tissues/cells to remain wet.

How long should I incubate my primary antibody?

  • Incubation for too short will not produce adequate signal. Incubation for too long can result in negative (unspecific) staining. Since the final result of your technique can only be determined at the end of your technique when no corrections can be made, the dilution and time of incubation for each antibody should be determined individually. In general, antibodies with known high affinity should be used at high dilution and overnight incubations. Antibodies with various affinities (polyclonal) must be experimented with to determine optimum time and dilution.

Problems with high background?

  • If you experience an overabundance of signal, try decreasing the primary antibody concentration and/or incubating for a shorter time.
  • Increase the blocking time or change your blocking solution.
  • If using an amplification staining strategy, reduce incubation time or concentration of reagents. 

No staining at all?

  • If you experience an absence of signal, try increasing the primary antibody concentration and/or incubating for a longer time.
  • Performing a western blot can confirm the activity of the primary antibody.  Remember to include positive controls.
  • Formalin and Paraformaldehyde fixative solutions can mask the antigen epitope.  Try a different antigen retrieval method ihcworld.com.

Non-specific staining?

  • Compare staining against negative control cells/sections (KO animal).
  • Run a western blot with the same sample you are staining for the presence of an monospecific band.
  • Do not let your cells/sections dry out.

 

IHC / ICC Protocols:

IHC / ICC Protocol: See 'Protocols and Procedures'

Additional references:

  • See product datasheet or contact Aviva Systems Biology

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Enzyme-Linked ImmunoSorbent Assay (ELISA)

General Tips to concider when performing an ELISA:

  • Increase antigen availability by building scaffold complex (1st streptavidin 4C overnight, then biotinylated antigen for 15min at RT).
  • Ensure proper positive and negative controls are performed.
  • Increased washing will decrease possible cross-reactivity between your reagents.
  • Use a detection strategy that will not cross-react with your sample.
  • Performing a western blot can confirm the activity of the primary antibody.  Remember to include positive controls.

Positive signal seen in negative control wells:

  • This may indicate a contamination of ELISA reagents or the samples themselves. Make sure your samples are not splashing into one another. Use known fresh reagents and perform ELIISA steps carefully.
  • If performing a sandwich ELISA - detection antibody may be binding to plating/coating antibody.
  • Check that all wells are being washed thoroughly with correct buffer during and after wash steps.
  • Ensure that you are using recommended amount of plating/coating and detection antibody. Try using less of either one to reduce signal.

High background seen across entire plate:

    • Detection antibody used in wrong amount/concentration or left on too long. Check that antibodies are being used in the recommended amount/concentration. Add stop solution when desirable level of signal is seen.
    • Check that substrate solution and stop solution are fresh. Fresh stop solution should be clear (yellow = contamination).
    • Make sure that stop solution has been added. Color will continue to develop if stop solution is not added.
    • Do not leave plate too long before reading. Color will continue to develop even after stop solution is added (stop solution slows color formation).
    • Ensure that all laboratory glasswares are clean and sterile.
    • Substrate incubation should be performed in the dark. Do not perform in the light. This will affect signal formation.
    • Check that incubators/thermometers are accurate and working. High temperatures can affect antibody binding kinetics. Incubation temperatures may need optimization.
    • Detection antibody is binding non-specifically. Check to make sure the recommended blocking step with an approved blocking buffer is included. When possible, use pre-absorbed, affinity purified antibodies.

see also suggestions under 'Positive signal seen in negative control wells'

Low signal seen:

  • Absence or low level of target protein expressed. Check that your target protein is expressed in your samples. Increase amount of sample used or change to more sensitive assay. Check that signal from positive control is within assay limits.
  • Too little antibody used. Check that the recommended amount/concentration of each antibody is used. This may require optimization.
  • Substrate solution is not fresh or improperly prepared. Prepare substrate solution immediately before each use. Ensure all solutions are prepared correctly, have not expired and have been stored properly.
  • Too short of an incubation time. Ensure you are following recommended incubation times. This may require optimization.
  • Antibody incubation temperatures are too low. Lower than optimum incubation temperatures can affect antibody binding kinetics. Check that incubators/thermometers are accurate and working. This may require some optimization. Ensure all reagents are at room temperature before beginning.
  • Stop solution was not added. Stop solution slows and stabilizes signal formation.

High signal seen in samples or positive controls, or signal does not decrease over a dilution range:

  • The concentration/amount of samples or positive controls is too high and out of range of the assay. Dilute samples and controls to within assay range.

Atypical signals seen across plate:

  • Confirm that plates are always on level surface to ensure even reagent and temperature distribution.
  • Ensure you are carefully using calibrated pipettes for all pipetting. Duplicates will ensure final signal accuracy.
  • Carefully mix all solutions/reagents before using.
  • If recommended, cover plates with proper lids during incubations. Do not let wells dry out. Maintain appropriate incubator humidity levels.
  • Make sure all wells are being washed correctly and uniformly.
  • Keep bottom of plate clean of any possible contaminants that might affect spectrophotometer.

Signal color developing too slowly:

  • Confirm plates and reagents are at room temperature before beginning.
  • Make sure you are preparing substrate solutions immediately before use. Ensure that all solutions are prepared correctly, have not expired, have been stored properly and are being used at the recommended amounts/concentrations.
  • The presence of contaminants like sodium azide and peroxidase can affect the substrate reaction.

Duplicate/Triplicate wells not yielding similar results:

  • Insufficient washing: If using an automatic plate washer, check that all ports are clean and free of obstructions, add a 30 second soak step and rotate plate halfway through the wash
  • Make sure you are using an ELISA plate and not a tissue culture plate
  • Do not reuse plate sealers
  • Your buffers may be contaminated.  Remake them.

Poor assay to assay variability:

  • This may be insufficient washing.  If using an automatic plate washer, check that all ports are clean and free of obstructions
  • Adhere to the recommended incubation temperature range for a given assay.  Avoid environments where temperatures vary
  • Follow the same protocol from assay to assay
  • Use fresh plate sealers for every assay
  • Check your standard curve dilution calculations and make a new standard curve
  • Use internal controls
  • Remake fresh buffers

If edge effects are being experienced, try:

  • Use fresh plate sealers
  • Avoid incubating plates in environments where temperatures vary

If drift effects are being experienced, try:

  • Making sure that all reagents are at room temperatures before pipetting into the wells unless otherwise instructed by the assay literature
  • Always perform an assay continuosly; have all standards and samples ready to go when protocol instructs you to use them

 

ELISA Protocols:

ELISA Protocol: See 'Protocols and Procedures'

Additional references:

  • See product datasheet or contact Aviva Systems Biology

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Immunoprecipitation (IP)

High Background:

  • Beads are not pre-blocked well enough with BSA. Make sure the BSA is fresh and incubate fresh beads 1 hour with 1% BSA in PBS. Wash 3-4 times in PBS before using them.
  • Using too much antibody or too much cell lysate can cause non-specific binding.
  • Exposing the cell lysate to empty beads, or beads bound by antibody of a different specificity, can remove non-specific proteins.

No target protein in eluate:

  • Ensure that the protein of interest is expressed in cells/tissue used.  If expressed at low amounts, increase the amount of cell lysate used.
  • Check to make sure you are using the correct beads to capture the specific antibody isotype.
  • Check to make sure your elution buffer is of the correct salt concentration and pH to break up the antigen:antibody complex.

How much antibody should I use for my immunoprecipitation reaction?

  • In general, 2-10 ug of antibody are required per 500 ug of cell/tissue lysate.  An optimum antibody amount should be determined empirically.

I want to use an IgM antibody for an immunoprecipitation.  How do I do this?

  • As IgG antibodies don’t bind Protein A/G, you can use Protein L, or Protein A/G beads that have a conjugated anti-IgM antibody.

Why does my IP reaction appear as a slurry and not a pellet after centrifugation?

  • The IP reaction should be spun at speeds between 200xg to 500xg.  Higher speeds will break the protein A/G beads causing the slurry appearance.

Do I need to add protease inhibitors to my lysis buffer used for the washing steps?

  • No.

How can I avoid the appearance of the heavy and light chain bands on my post-IP material western blot?

  • To reduce the presence of your immunoprecipitation antibody on your western blot, it is recommended to cross link the antibody to a stable support, like beads.  The target protein can then be eluted with a mildly acidic eluent buffer, like a glycine-HCl buffer.

 

IP Protocols:

IP Protocol: See 'Protocols and Procedures'

Additional references:

  • See product datasheet or contact Aviva Systems Biology

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