Immunohistochemistry Antibody Validation Report for Anti-CD68 Antibody (STJ96952)
Presentation of Simon Glerup in 1st International Antibody Validation Forum 2014
-
Upload
st-johns-laboratory -
Category
Science
-
view
602 -
download
0
description
Transcript of Presentation of Simon Glerup in 1st International Antibody Validation Forum 2014
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…