This document provides information on an immunoprecipitation and chromatin immunoprecipitation workshop. It discusses the differences between IP, Co-IP, ChIP, and RIP techniques. It also outlines considerations for performing IP and ChIP experiments such as antibody choice, sample preparation, crosslinking, sonication, washing buffers, and troubleshooting tips. The workshop covers both theoretical background and practical protocols for IP and ChIP.

1. IMMUNOPRECIPITATION &
CHROMATIN IMMUNOPRECIPITATION (ChIP)
WORKSHOP

2. Bachelor in Pharmaceutical Chemistry from Jagiellonian University, Kraków, Poland
Master in Biotechnology from the University of Amsterdam, The Netherlands
M. Curie network – an International PhD student at the University of Barcelona, Spain
M. Curie network – a self-directed researcher and project manager, Biotalentum, Budapest, Hungary
Main research focus related to Central Nervous System and Oncological Disease Models
12 PubMed Publications, Szczesna K
Szczesna et al., 2018 May Cell Reports: Inhibition of Gsk3b Reduces Nfkb1 Signaling and Rescues
Synaptic Activity to Improve the Rett Syndrome Phenotype in Mecp2-Knockout Mice
Dr. Karolina Szczesna

3. FROM OUR BENCH
TO YOUR BENCH™
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• Antibodies (mostly full-length protein)
• ELISA Kits
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4. Dr. Karolina Szczesna
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5. Dr. Karolina Szczesna

6. 1
PART 1: IMMUNOPRECIPITATION (IP)
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7. OVERVIEW
• IP, Co-IP, ChIP, RIP– What is the difference?
• IP applications
• Considerations before starting IP
• IP protocol
• IP troubleshooting
OVERVIEW

8. Different types of immunoprecipitation:
• IP to isolate a single protein
• Co-IP to study the interactions between the primary
antigen protein and other proteins
• ChIP to study the interactions between the primary
antigen protein and DNA
• RIP to study the interactions between the primary
antigen protein and RNA.
Dr. Karolina Szczesna

9. Different types of immunoprecipitation:
• IP to isolate a single protein
• Co-IP to study the interactions between the primary
antigen protein and other proteins
• ChIP to study the interactions between the primary
antigen protein and DNA
• RIP to study the interactions between the primary
antigen protein and RNA.
Dr. Karolina Szczesna

10. APPLICATIONS
–
Isolation and detection of protein-of-interest
–
Enrichment of low-expressed proteins (not possible by WB)
–
Investigation of protein–protein interactions
– Identification of proteins as part of a protein complex
Dr. Karolina Szczesna
APPLICATIONS

11. Immunoprecipitation (IP)
§ IP is a technique used to purify and enrich the protein-of-interest out of a
protein mixture.
§ IP-isolated proteins can then be further analyzed by Western blotting,
ELISA, and mass spectrometry.
IP helps to identify: presence,
up/down
regulation, size, stability, and
interactions of the protein-of-interest.
Dr. Karolina Szczesna
IMMUNOPRECIPITATION (IP)

12. IMMUNOPRECIPITATION (IP)
– IP based on a solid phase (bead) –
contains a binding protein.
– IP requires a specific antibody to purify
a single antigen.
– The sample containing the protein-
of-interest is incubated with the beads
and antibody.
– The antibody binds to the protein
and beads.
– The beads are washed.
– The protein is eluted.
Dr. Karolina Szczesna
IMMUNOPRECIPITATION (IP)

13. CONSIDERATIONS BEFORE STARTING IP

14. Main Factors Possible Solution
Wash, elution, binding buffer Composition, volume
Antibody Amount, specificity
Pre-clearing of lysate Non-specific binding
.
– Most important:
the purification of a specific protein with specific binding to the
antibody.
Dr. Karolina Szczesna
VARIABLE FACTORS DEPEND ON THE
PROTEIN-OF-INTEREST AND THE ANTIBODY

15. • Protein A & G are suitable for the detection of IP proteins: binding
to the intact Fc regions of IgGs.
• Protein A/G binds to all subclasses of immunoglobulins
There are three types of beads available:
– Agarose (Sepharose) beads
– Magnetic beads (commonly used)
– Nanotrap (new! low background)
BINDING PROTEINS & BEADS

16. Affinity of Binding Proteins:
different immunoglobulin subclasses
Binding Capacity
++++ Strong Binding
+++/++ Medium Binding
Variable/– Weak or No Binding
Dr. Karolina Szczesna

17. In IP, the target protein is usually in the na ve conforma on.
Ø an odies used for IP need to recognize and have high
affinity to the epitope on the surface of the protein.
Ø What to consider before choosing the right an ody:
ü The immunogen is on the exposed surface of the protein (immunogen).
ü The an dy works for ChIP and/or IHC.
• Monoclonal an recognize a single epitope
• Polyclonal an odies recognize several epitopes
Tip: Where possible, use a polyclonal an body to capture the target protein.
Polyclonals bind to mul ple epitopes on the target protein, meaning a greater amount
of protein is retained.
CHOOSING THE RIGHT PRIMARY ANTIBODY

18. • a capture antibody from one species,
and an antibody for WB detection
from another
• ensure that both antibodies recognize
different epitopes of the target
protein, in addition to originating
from different species
Tip: use a combination of a polyclonal
(capture) antibody and a monoclonal
(detection) antibody
This combination ensures maximum capture
efficiency with high detection specificity
ANTIBODY PAIRS
POLYCLONAL OR MONOCLONAL

19. Dr. Karolina Szczesna
ANTIBODY TITRATION FOR IP ISOTYPE & NEGATIVE CONTROL
BANDS
• Titration should range from 1 to
10.0 µ g for ~5000 µ g of protein
extract.
Example
• 1:100 dilution
• the titration experiment
• To keep the same experimental conditions,
after selecting the incubation time, all
dilutions should be performed with the
same type of sample.
• Isotype Control
An isotype control is used to determine
which bands in the experimental sample are
specific versus non specific signal due to
the isotype.
• Negative Control
Plain beads (without antibody) can be used
as negative controls. They help to
distinguish specific and non specific
bindings.

20. IP PROTOCOL OVERVIEW

21. – Prepare sample lysate: RIPA buffer
– Commonly used amounts: 0.3–0.5 ml lysate containing 1–4 mg
total protein
– Use protease inhibitors
Sample Preparation
Tips
– High concentrations of detergent interfere with immunoprecipitation
Ø Try to lyse cells with a small volume of RIPA and then dilute the
lysates with PBS to the final volume.
– Use sufficient lysates for the first trial. Typically 1–3 mg total
protein is needed for each IP.
– The concentration of proteinase inhibitor should be 1.5–2 times
that for Western blotting lysates.
Dr. Karolina Szczesna

22. - resuspend Protein A/G sepharose beads slurry (gentle Vortex)
- add 50 μl of 50% beads slurry per 0.5–1 mg of cell lysate
- incubate at 4°C for 30 min on a rotator
- centrifuge at 1000 rpm for 3 min at 4°C
- transfer the supernatant to a fresh tube.
Lysate Pre-clearing
Tip
Tissues with abundant IgG are suggested to be pre-cleared with Protein A or G sepharose.
Dr. Karolina Szczesna
LYSATE PRE-CLEARING

23. SAMPLES PRE-CLEARING
Dr. Karolina Szczesna
SAMPLES PRE-CLEANING
BEFORE THE ACTUAL IP EXPERIMENT
. Helps to remove unwanted proteins to the solid support
. The sample is incubated with the plain beads
. Non specific components that bind to the solid phase will be removed

24. Immunoprecipitation
Tips Do not use too much Protein A or G sepharose
Protein A or G will bind IgG in Western blotting and cause intense non-specific bands.
ü Add appropriate amount of primary antibody to the whole
(or pre-cleared) lysate. Titration experiment.
ü Gently rock the mixture at 4°C for 2–4 h or overnight.
ü Set up a negative control (IgG corresponding to the primary antibody source).
ü Add Protein A or G sepharose beads. Gently rock the mixture at 4°C for 1–4 h.
ü Centrifuge the mixture at 500–1000 rpm for 30 s at 4°C and
discard the supernatant.
ü Wash the beads 3–4 times with 1 ml RIPA lysis buffer or 1X PBS
with 0.2% Tween 20 (less stringent), centrifuge and discard
the supernatant.
Carry out pre-equilibration of beads in the buffer
Beads are usually stored in solution containing, e.g., ethanol that would interfere with the binding.
Equilibrate - 2–3 washings
Do not spin beads at max speed
May destroy/decompose beads
Dr. Karolina Szczesna

25. Elution
Tip
Increasing the salt concentration in the elution buffer will help to elute protein.
ü Elute the pellet twice with 40 µl 0.10 M Glycine, 0.05M
Tris-HCl (pH 1.5–2.5) elution buffer containing 500 mM
NaCl.
ü Pool elutions and neutralize by Alkali neutralization
Buffer or 10X PBS buffer (pH 6.8–7.2) to a final of 1X.
ü Add 5X SDS sample buffer to the elutions. Heat at 95°C
for 5 min.
For low concentrated samples: after elution it is possible to concentrate samples.
Use Trichloroacetic Acid (TCA) precipitation protocol before addition of SDS sample buffer.
Dr. Karolina Szczesna
ELUTION

26. Binding Buffer
Washing Buffer
– Some bindings to protein A or G can be enhanced by adapting
the pH value (e.g., Protein G binds best to IgG at pH 5.0).
The washing step should not interfere with the desired protein bindings.
The washing step should remove all unwanted protein bindings and debris.
Tip: Use gravitational flow through filter columns loaded in microfuge tubes.
If the observed background signal is too high in an IP
experiment, the buffer type and additives can be varied.
Commonly used buffers are PBS or TBS.
– Reagents: NP40, Triton-X, CHAPS.
– Additives: DTT (reduction of disulfide bonds).
Dr. Karolina Szczesna
BUFFER OPTIMIZATION TIPS

27. Elution Buffer
– If the IP sample is further used for Western blotting, the
sample can be directly diluted in SDS-PAGE sample buffer
containing reducing agents.
– Most commonly used elution buffer: glycine 0.1 M at pH 2.5–3.5 and
1% of SDS to disturb the bead–antibody–antigen interactions.
– If the antibody-protein binding does not dissociate or if the
protein gets denatured, the pH can be changed.
Please note: Denatured Proteins Protocol Modifications
where the epitope of interest may not be accessible in the native conformation
Dr. Karolina Szczesna
BUFFER OPTIMIZATION TIPS

28. IP TROUBLESHOOTING

29. Dr. Karolina Szczesna
TROUBLESHOOTING

30. Dr. Karolina Szczesna
TROUBLESHOOTING

31. Dr. Karolina Szczesna
TROUBLESHOOTING

32. 1
PART 2: CHROMATIN IMMUNOPRECIPITATION (ChIP)
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33. OVERVIEW
• ChIP applications
• Considerations before starting ChIP
• ChIP troubleshooting and tips
• Helpful resources
OVERVIEW

34. – Epigenetics provides a live cell picture of the native chromatin structure
– detect special protein–DNA interactions
– monitors transcriptional regulation via histones modifications
– further analyzed by qPCR or sequencing (ChIP-seq)
– ChIP provides a live cell picture of
the native chromatin structure and
factors bound to genes in different
functional states
– ChIP methodology involves protein–DNA cross-linking
– crude chromatin is sonicated to small fragments,
usually with an average size of 300–1000 bp
Dr. Karolina Szczesna
CHROMATIN IMMUNOPRECIPITATION (ChIP)

35. Main Steps:
crosslinking
cell lysis
chromatin preparation IP
Reverse crosslinking
DNA clearing
DNA quantitation
Dr. Karolina Szczesna
ChIP OVERVIEW

36. CONSIDERATIONS BEFORE STARTING ChIP

37. ChIP & Cell Line Samples
• Ensure the appropriate number are
resuspended in 10 ml of fresh media.
• Add formaldehyde to reach a final
concentration of 1%.
• Vortex for a few seconds.
• Incubate for 10 min at RT in agitation.
ChIP & Tissue Samples
• Grind frozen tissue into powder.
• Pour the resulting powder into a 15 or
50 ml falcon.
• Add formaldehyde to reach a final
concentration of 1%.
• Vortex for a few seconds.
• Incubate for 10 min at RT in agitation
Dr. Karolina Szczesna
CHROMATIN PREPERATION FROM CELLS & TISSUES

38. ChIP Tip 1:
ChIP Tip 2:
Always keep cells/tissue on ice
– Temperature is critical.
Perform cell lysis at 4°C.
Keep the samples ice-cold and use ice-cold buffers.
Under/over cross-linking
Please note: When using paraformaldehyde, ensure that
it is freshly prepared (final concentration of 1%–1.5%).
– Under cross-linking can prevent the disassociation
of protein–DNA complexes and result in poor yield.
– Over cross-linking can mask epitope sites crucial
for antibody binding, prevent chromatin shearing,
and inhibit the successful uncross-linking of the
protein–DNA complex.
Dr. Karolina Szczesna

39. ChIP Tip 3: Chromatin shearing and sonication
– Avoid large fragments in the tissue suspension.
– Pipette with cut tips for better homogenization.
– Ensure the sonicator probe is not in contact with the tube wall.
– Increase the number of sonication steps:
however, avoid increasing the time (or the power) of each step as this
may overheat the sample and lead to loss of antigenicity.
– Add ice to the sonicator to prevent the sample overheating.
Dr. Karolina Szczesna

40. ChIP Tip 4: Bead and primary antibody choice
Beads
– Always fully resuspend beads by vortexing before pipetting.
– Always store at 4°C and never allow beads to dry out.
– Check the subclass of your antibody is compatible with Protein A/G.
Antibody
– Insufficient amount of antibody can result in not enough material
for successful PCR analysis.
– Too much antibody can increase PCR background.
Dr. Karolina Szczesna

41. ChIP Tip 4: Negative ChIP controls
– Use non-immune IgG in the IP incubation mix from
the same species the antibodies were produced in.
– Incubate IP fraction with beads (without antibody coating).
Dr. Karolina Szczesna

42. ChIP Tip 5:
ChIP Tip 6:
IP efficiency in reverse cross-linking
– 15-minute incubation at 95°C
Some samples require Proteinase K treatment for a few hours at 65°C.
DNA elution and purification
– Use different washing buffers (low & high salt, LiCl and TE buffers).
– While using a commercial purification column, check the column is
completely dry after the washing step as any leftover will inhibit
elution.
– Make sure the elution buffer is placed directly onto the silica membrane
and allowed to adsorb for at least one minute.
Dr. Karolina Szczesna

43. ChIP Tip 7: Analysis on Immunoprecipitated DNA
– To avoid variations between replicates, add the same
amount of beads for all samples.
– Complete the elution of chromatin from beads.
Elution is optimal at 65°C with frequent mixing to keep beads
suspended in solution (~10 minutes).
Please note: A weak PCR signal or no DNA amplification shown in the samples may be due to an
inadequate primer result in the PCR amplified region spanning the nucleosome-free region.
Dr. Karolina Szczesna

44. ChIP TROUBLESHOOTING & TIPS

45. 27/11/12 Half brain
ChIP
10 min Crosslink
28/11/12 Half brain
ChIP
8 min Crosslink
Tip: 10-minute incubation at room temperature in agitation.
Dr. Karolina Szczesna

46. 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
5ul 15ul
Sample volume
Tip: 10–15 ul per well is usually enough.
Dr. Karolina Szczesna

47. 1
FC H
2
FC H
3
FC
4
FC H
5
FC
6
FC H Whole brain
Sonication
5 min
17 min
Tip: 15 minutes (high power; 30 seconds sonication, 30 seconds rest).
Dr. Karolina Szczesna

48. S153 R153
30’ sonication 45’ sonication
S153 R153
Dr. Karolina Szczesna

49. Dr. Karolina Szczesna
TIPS BEFORE STARTING ANALYSIS ON IMMUNOPRECIPITATION DNA
(PCR AMPLIFICATION)
1. Prepare new solutions from stocks to avoid contamination.
2. Test primer pairs for genomic DNA using different primer conditions and dilutions.
The use of standard/input DNA will determine the efficacy of the primers.
3. Ensure there is enough DNA in the PCR reaction and your PCR conditions are optimal.
Add more DNA to the PCR reaction or increase the number of amplification cycles if needed.
4. To avoid variations between replicates, add the same amount of beads for all samples.
Ensure beads are well suspended while pipetting.
5. While there is no product in the positive control ensure there is enough chromatin or
antibody within the IP reaction.
6. Complete the elution of chromatin from beads.Elution is optimal at 65 C with frequent mixing
to keep beads suspended in solution (~10minutes).

50. HELPFUL RESOURCES

51. Dr. Karolina Szczesna
MONOCLONAL OR POLYCLONAL

52. PaxDb: Protein Abundance Database:
http://pax-db.org
STRING database: Assessment and integration of
protein–protein interactions:
https://string-db.org/cgi/input.pl
UniProt: Universal Protein resource:
http://www.uniprot.org/uniprot/
NCBI-BLAST: Online sequence alignment:
https://blast.ncbi.nlm.nih.gov/Blast.cgi
GeneCards: genomic, proteomic, transcriptomic,
genetic, and functional information database:
http://www.genecards.org
PubMed: most up-to-date information regarding target research:
https://www.ncbi.nlm.nih.gov/pubmed
Proteintech: free protocol guides:
https://ptglab.com/promotions/free-protocol-guides/
Dr. Karolina Szczesna
USEFUL LINKS

53. • The preservation of protein post-
translational modifications (PTMs)
plays an essential role in regulating
chromatin dynamics
• Maintaining PTMs during cell lysis
is important for in vivo interactions
in vitro
PHOSPHATASE, METHYLATION & DEUBIQUITINA
Dr. Karolina Szczesna
PHOSPHATASE, METHYLATION & DEUBIQUITINATION INHIBITORS

54. • The preservation of protein post-
translational modifications (PTMs)
plays an essential role in regulating
chromatin dynamics
• Maintaining PTMs during cell lysis
is important for in vivo interactions
in vitro
Example: adding phosphatase
inhibitors to the lysis buffer will
maintain the phosphorylated stage of
the protein.
PHOSPHATASE, METHYLATION & DEUBIQUITINATION
Dr. Karolina Szczesna
PHOSPHATASE, METHYLATION & DEUBIQUITINATION INHIBITORS

55. § Antibodies against 12,000 targets, all manufactured in-
house
§ Open access validation data on the website
§ First antibody company to use KO/KD to demonstrate
specificity
(The CiteAb awards for the very best suppliers and individuals in the antibody industry)
Dr. Karolina Szczesna

56. § Antibodies against 12,000 targets, all manufactured in-
house
§ Open access validation data on the website
§ First antibody company to use KO/KD to demonstrate
specificity
(The CiteAb awards for the very best suppliers and individuals in the antibody industry)
Dr. Karolina Szczesna

57. CiteAb 2017:
“‘Antibody supplier to watch in 2018”
recognizing the company with the largest percentage increase in citations based on data
gathered between January 2017 and December 2017.
CiteAb 2018:
“‘Rising star’ in immunology”
recognizing the company with the largest increase in citations in immunology over previous years.
957
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3267
4187
5967
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The trend of publication in every year
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The trend of total publication
Dr. Karolina Szczesna
PROTEINTECH IN PubMed

58. CONTACT US
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59. Dr. Karolina Szczesna
Senior Product Manager
& Technical Support
THANKS YOU FOR YOUR ATTENTION