Immunoprecipitation Workflow and Troubleshooting Tips

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Immunoprecipitation Workflow and Troubleshooting
Tips
CD
Immunoprecipitation (IP) Work?ow Pre-clearing
Protein Lysate
Cell Lysis
Antibody
Protein A Magnetic Beads
Binding
Washing
Protein of Interest
Elution
Cell lysis The lysis step for IP should be
relatively gentle to allow antibody-antigen
binding, but also harsh enough to ef?ciently
extract proteins from the cells. Thus, it's
important to choose a lysis reagent that can
guarantee high protein yields and retain protein
activity.
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• Pre-clearing This step reduces the number of
  non-speci?c contaminants in the cell lysate as
  well as removes proteins that possess a higher
  af?nity to protein G or protein A before the
  speci?c IP steps. As a result of pre-cleaning,
  there would be less background noise and an
  improved signal-to-noise ratio.
• Binding Magnetic beads (or agarose), antibody,
  and antigen form complexes at this step. The
  buffers used here and at the washing steps are
  vital to perform a successful IP. The order that
  these three components are added also affect the
  results. Antibodies may be added ?rst to
  covalently or noncovalently bind to the magnetic
  beads before adding lysate, or incubated with
  lysate to form a complex before adding protein A
  or protein G magnetic beads to purify the complex
  from the mixture.
• Washing Ideally, this step can break all
  nonspeci?c bindings without affecting the speci?c
  interactions between antibody and antigen.
  Adding extra lysis buffer to the washing buffer
  can help breaking the nonspeci?c bindings.
• Elution Generally, there are three kinds of
  elution buffers, including glycine, SDS, and urea
  elution. Any of the three elution buffers can be
  used to elute the protein from the beads. The SDS
  buffer is harsh enough to elute noncovalently
  bound antibodies, antibody fragments, and the
  protein of interest. The glycine buffer is a
  gentler choice.
• Troubleshooting Tips
• 1. To improve elution conditions
• Choose an appropriate lysis buffer according to
  the protein location (membrane, cytosolic, or
  nucleus). The pH, detergent, salt, and divalent
  cation concentrations should be optimized for
  each IP.
• Check the antibody binding properties of each
  beads. Its important to match the binding
  speci?city of the beads to the species and the
  antibody subtypes (Table 1).

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Table 1. Immunoglobulin binding
properties. Protein A Protein G
- - - - - - - -
- - - -
- -

Species Immunoglobulin Isotype
Protein A/G
Human IgG1

Human IgG2

Human IgG3

Human IgG4

Human IgM

Human IgE

Human IgD
-
Human IgA

Human IgA1

Human IgA2

Human Fab

Human ScFv

Mouse IgG1

Mouse IgG2a

Mouse IgG2b

Mouse IgG3

Mouse IgM
-
Rat IgG1

Rat IgG2a

Rat IgG2b

Rat IgG2c

Cow IgG1

Cow IgG2

Sheep IgG1

Sheep IgG2

Goat IgG1

Goat IgG2

Chicken IgY
-
Hamster IgG

Pig IgG

Horse IgG

Rabbit IgG

Cat IgG

Rhesus Monkey IgG

Strong binding Medium binding Weak
binding - No binding.
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• Alter the components, salt concentration, or pH
  of the elution buffer if no protein of interest
  is eluted from the beads.
• Conduct a titration experiment ?rst to optimize
  the antibody concentration if there isnt enough
  antibodies for proper binding.
• To enhance the expression of protein of interest,
  increase the volume of the cell lysate and
  pre-clearing the sample to decrease non-speci?c
  binding and remove debris.
• Polyclonal antibodies usually perform better than
  monoclonal antibodies.
• Spin lysate for 30 min before adding the
  antibody. This can remove insoluble proteins,
  membrane fragments, and debris to reduce the
  number of the competing proteins in the sample.
• Primary antibody and antigen of interest can be
  incubated from 4 hours to overnight at 4ºC.
• Avoid using lysates containing substances such as
  dithiothreitol, 2-mercaptoethanol, or other reduc
  ing agents. This will affect antibody binding.
  Extremes in pH and excessive detergent
  concentrations also affect antibody-antigen
  interaction.
• 2. High background
• Remove supernatant immediately after
  centrifugations to avoid carryover of
  detergent-insoluble proteins.
• To thoroughly wash the samples, place a lid on
  the tube and invert several times before
  centrifugation.
• Pre-blocking beads with fresh BSA can decrease
  non-speci?c protein binding to the beads. Beads
  are incubated with 1 BSA in PBS for an hour and
  then wash 3 to 4 times in PBS before use.
• Use an af?nity-puri?ed antibody with high
  speci?city to avoid high background.
• Use recommended numbers of antibody or optimize
  the concentration of the antibody by titration.
  Using too much antibody may cause non-speci?c
  binding.
• Decrease cell numbers to avoid high protein
  concentration in the lysate.
• In case of too much non-speci?c binding of
  proteins to antibody, use an irrelevant antibody
  of the same species of origin and the same Ig
  subclass to pre-clear the lysate.
• Add fresh protease inhibitors in the sample
  lysate to prevent antigen degrading during IP.
• Inappropriate washing may cause high background.
  Use distilled water and increase the number of
  washes. Before the last wash, it is a good
  practice to transfer the cell pellet to a new
  tube.

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4. Antibody heavy/light chains background If the
target protein is about 25kDa or 50kDa, the
detection signal will be masked by the antibody
chain. There are two solutions to this problem
1) use capture and detection antibody originated
from different species 2) crosslink the capture
antibody with protein beads and use these
conjugates to precipitate the protein of
interest, if using antibodies from the same
species. Creative Diagnostics provides a
comprehensive list of af?nity magnetic particles
for immunoprecipitation and protein separation,
including protein A and protein G magnetic
particles in wide range of sizes. Please visit
our website to see more.
For more information, view our website
www.cd-bioparticles.com
Email info_at_cd-bioparticles.com
Tel [phone] Fax [phone]
Address 45-1 Ramsey Road, Shirley, NY 11967, USA
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