Embed Size (px)
DESCRIPTION
Dr. Glerup is Associate Professor at Aarhus University, Denmark and Mayo Clinic, Florida. He earned his PhD in molecular biology at Aarhus University in 2008 and continued as postdoc in neuroscience where after he was appointed group leader in 2012. His laboratory focusses on molecular pathways in cognition and employs a variety of techniques including transgenic animal models, molecular neurobiology, and imaging. The laboratory is particular interested in a family of sorting receptors, sortilins, and their role in regulating neuronal development and function, and further how they contribute to the development of mental disorders. Dr. Glerup is also the founder of the antibody review database pAbmAbs (www.pabmabs.com) that collects information on the use of antibodies in research. For more details about 1st international antibody validation forum please check on http://www.stjohnslabs.com/ac_cms/blog
Citation preview
Simon Glerup, Aarhus University
Navigating the good, the bad and the ugly
research antibody
Usefulness of antibody review platforms
A ”good” antibody can detect a single protein in a complex mixture
Antibodies are fundamental tools in medicine and life science
Finding antibodies is like looking for a five-star hotel in London
This is what you are promised
Sometimes this is what you get…
How proteins control neuronal function and influence behavior?
Place preference test
Mice can learn to prefer an alcohol-associated context
SorCS2 knockouts show no alcohol-induced place preference
SorCS2 knockouts show no alcohol-induced place preference
SorCS2 knockouts react exagerated to alcohol
Interneuron markers
The SorCS2 gene is critical for memory formation in mice
NMDA receptor antibodies
1. AGC-003
2. BD610416
4% PFA/Methanol
HEK293 cells transfected with:
EGFP-GluN1
EGFP-GluN2B
(Forms functional NMDAR complex)
4% PFA Methanol
Ab: BD610416, GluN2BHippocampal neurons DIV14 (WT)
MAP2 (red)
GluN2B
GluN2B
TrkB antibodies
TrkB is a key protein in the development and function of the nervous system
Product Manufacturer Price (DKK) Works as promised?
610102 BD Biosciences
~3000 No
TrkB pAb Cell Signalling ~3000 No
TrkB mAb Cell Signalling ~3000 No
sc-8316 Santa Cruz ~2000 No
sc-11 Santa Cruz ~2000 No
sc-139 Santa Cruz ~2000 No
sc-414 Santa Cruz ~2000 No
sc-12 Santa Cruz ~2000 No
Anti-TrkB Lou Reichardt - No
AF1494 R&D Systems ~3000 Excellent
How could we increase transparency in research?
What do the researchers think?
Low-Density Lipoprotein (LDL) receptor family
members
• Human LRP1 and LRP2
have a sequence identity
of ~40%
Bu G., Nature Reviews Neuroscience 10, 333-344 (2009)
• Heavily expressed in kidney proximal tubules
• Involved in tubular reabsorption of proteins
Megalin
LRP2 expression in the kidney
Christensen EI. et al. Physiology 27:223-236, 2012
The Human Protein Atlas Project
LRP1 IHC stain with
antibody HPA022903
in human kidney biopsy
=> strong proximal tubule stain
Antibody HPA022903
Antibody specificity analysis for HPA022903 :
Western blotting : Supportive - Band of predicted size in kDa.
Protein arrays : Supportive - Pass with single peak corresponding to interaction only with its own antigen.
Testing Antibody HPA022903 in LRP2 knockout mice by IHC:(data provided by pAbmAbs reviewer Kathrin Weyer)
Conclusion: The Ab recognizes LRP2, not LRP1, in the kidney proximal tubule cells
Wild type LRP2 Knockout
Conclusions
Success with research antibodies depends on:
The person carrying out the experiment
Subtle experimental details (fixation, incubation, concentration, blocking)
Type of tissue – cell line – expression levels
Quality of equipment – e.g. sensitivity of confocal microscope
Antibody batch
----------------------------------------------------------------------
The best advice you can get is still that of a trusted colleague
Thanks to all the reviewers…
