1. Blame it on the antibodies: the most common problems with antibodies and how to avoid them
What is the problem?
We have a reproducibility crisis in science. The consequence is a enormous waste in materials, time and money, costing an estimated US$350 million annually in the US alone!
‘Bad’ antibodies are considered part of the reproducibility crisis.

2. Blame it on the antibodies: the most common problems with antibodies and how to avoid them
What is a ‘bad’ antibody?

3. A few examples (worth a read):
Blame it on the antibodies: the most common problems with antibodies and how to avoid them
A few examples (worth a read):
In a 2008 study (Berglund et al. 2008, Mol. Cell. Proteomics) fewer than half of ~6.000 routinely used commercial antibodies recognized only their specific targets!
In a 2009 study on 49 commercially available antibodies targeting 19 different GPCRs (Michel et al. 2009, Arch. Pharmacol.), most bound to more than one target protein!
In 2011, an evaluation of 246 antibodies used in epigenetic studies found that one quarter failed tests for specificity (Egelhofer et al. 2011, Nat. Struct. Mol. Biol.)!

4. Blame it on the antibodies: the most common problems with antibodies and how to avoid them
What is the cause of the ‘bad’ antibody problem?
Three decades ago scientists had to make and validate their antibodies themselves.
Today, more than 300 companies sell over 200 million antibodies: a market worth 1.6$ billion!
Commercially available antibodies are often only poorly validated for target specificity and lot-to-lot variability!
Different vendors provide different levels of validation, depending on their approach to the balance between profit making and high quality provision

5. Blame it on the antibodies: the most common problems with antibodies and how to avoid them
Berglund et al. 2008, Mol. Cell. Proteomics

6. How should we validate an antibody?
Blame it on the antibodies: the most common problems with antibodies and how to avoid them
How should we validate an antibody?
‘Antibodies are not magic reagents. We can’t just throw them onto our sample and expect the result we get is 100% reliable without putting some critical thinking into it!’
Bordeaux et al. 2010, Biotechniques

7. Polyclonal vs. monoclonal antibodies
Polyclonal antibodies
-Purified from antiserum raised against antigen
-Heterogeneous mixture of different antibodies
Each Ab is a product of different plasma cells
Are immunologically different
React with various epitopes on the antigen
Less sensitive to variations in sample preparations
Better for detection of low antigen levels/fast capture (IP)/denatured proteins (WB)
Large amounts of non-specific antibodies
Cross reactivity/background possible in some applications
Easy and cheap to produce
Limited quantities available
Batch-to-batch variations are common
Monoclonal antibodies
Product of an individual clone of plasma cells
Are immunologically identical and have uniform affinity
React with a specific epitope on the antigen
Absence of nonspecific antibodies/low background
Better choice for cell/tissue staining and flow sorting
Targeted epitope must survive fixation
Expensive to produce/cell culture required
Long timeframe for hybridoma production
Unlimited quantities available
No batch-to-batch variability

8. How do we find a good antibody?
Blame it on the antibodies: the most common problems with antibodies and how to avoid them
How do we find a good antibody?
Know your biochemistry
Read, read, read: screen the available literature and the internet about the antigen of interest.
Which antibodies have already been used and validated in other studies?
Maybe someone has made an antibody themselves? (those are usually properly validated)
Browse databases:
Check out ‘reliable’ vendors that provide user-feedback reports and rankings, e.g Abcam,
Cell Signalling Tech, St. John’s Labs.
Check out the ‘developmental studies hybridoma bank (DSHB,
for monoclonal antibodies.
Read the datasheet: check if the antibody has been tested for the specific application
you need it for.

9. How to validate an antibody for western blotting?
Knock-down / knock-out negative controls
Positive controls (overexpression)
Activated/inhibitor-treated samples for signalling pathways
Walko et al. 2017, Nat. Comms
Ackerl et al. 2007, J. Cell Sci.
®CST

10. How to validate an antibody for immunofluorescence staining of cells?
Walko et al. 2017, Nat. Comms
®CST
AGR2
®CST
NFkb

11. Antibody dilution for optimal results
1:200
1:400
1:800
1:1.600
1:3.200
1:6.400
αKindlin2 / DAPI
Courtesy of E. Rognoni

12. How to validate an antibody for immunofluorescence staining of tissues?
Knock-down / knock-out negative controls
Rognoni et al. 2014, Nat. Med.

13. How to validate an antibody for immunofluorescence staining of tissues?
Blocking peptides/proteins
Lim et al. 2013, Science

14. How to validate an antibody for flow sorting?
Rabbit mAb IgG
CREB Rabbit mAb
HeLa (CD102neg.)
HUVEC (CD102pos.)
®CST

15. Fixation
Fixation is the preservation of cells and tissues in a reproducible and lifelike manner
What is it use for?
Fixatives stabilize the cells and tissues
Fixatives prevent autolysis by inactivating lysosomal enzymes and inhibit growth of bacteria
Fixation breaks down cell barrier and allows relatively large molecules to penetrate
Problems?
Fixatives denature proteins by coagulation, by forming additive compounds or by a combination of the two
The resulting complexes differ from the undenatured proteins in both chemical and antigenic profiles
 Fixation is always a compromise

16. PFA 1%
αKindlin1 / DAPI αPaxillin/ DAPI Overlay
Courtesy of E. Rognoni

17. MeOH:Acetone αKindlin1 / DAPI αPaxillin/ DAPI Overlay
Courtesy of E. Rognoni

18. Formaldehyde-based fixatives
Positive:
Fixatives are well tolerated by tissues and have good penetration
Formaldehyde reacts primarily with basic amino groups to form cross-linking “methylene bridges”
Formaldehydes do not permeabilize cells/tissues
Negative:
Antigen masking is very common
Proteolytic enzyme digestion and/or antigen retrieval often necessary

19. Methanol and acetone Simultaneous fixation and permeabilization
Remove lipids from the plasma membrane and coagulate (precipitate) the proteins
Positive:
Permit good antibody penetration
Often excellent preservation of antigens
Proteolytic digestion or antigen retrieval are not required
Negative:
Penetration in tissue is very poor
Fixation is often not complete
Sometimes the localization of soluble proteins is disrupted

20. Paraffin or Frozen sections?
Paraffin section Frozen section +
Morphology is better preserved
Better retention of labile proteins, nucleic acids and small peptides
Fixation and embedding cause antigen masking
Some epitopes do not survive fixation and embedding.
Enzymatic- or heat- mediated antigen retrieval must be used +
Antigen preservation is better
Preparation is faster
Selection of different and optimal fixative for each antigen possible all from the same block
Morphological detail and resolution of the frozen sections is usually inferior
Frozen sections are less stable + + + - - - - -