Dear Elite Researcher,
You know how a pulldown protocol (IP) can work for one protein but fail for another.
IPs are stubborn and highly individual.
However, Wildtype One found a few lesser-known tips that can enhance your protocol and help you avoid failures.
Here’s what to do before, during, and after your IP:
Tips for experiment design
IP Rule of Thumb - Always start with “1-1-40-1-4-3” then adjust based on your results:
+1 µg antibody
+1 mg protein lysate
+40 µL beads
+1 h incubation
+4 °C
+3 washes
Your ultimate “negative control” is not an isotype control (IgG), it’s knockout/knockdown lysate—do it on the side to confirm
Match your antibody’s species to the right beads:
-A for rabbit/human IgG1/2/4
-G for mouse IgG1/rat IgG
-L for κ light-chain Fabs/nanobodies
-Class-specific for IgM/IgA/IgY
Tips for before the IP
Block beads with BSA—it actually works
Pre-clear lysates with naked beads to remove sticky proteins
For chromatin, add salmon sperm DNA to reduce nonspecific binding
Add moderate salt (300–500 mM NaCl) and mild detergent (0.05–0.1% NP-40 or Tween-20) to all wash buffers
Match detergent strength to biology: gentle (NP-40, digitonin, DDM) for complexes, harsh (RIPA) for solubilization.
Add glycerol (5–10%) for fragile complexes
Add phosphatase inhibitors or pervanadate immediately when targeting phosphorylation states
Avoid EDTA or EGTA if pulling down calcium-sensitive interactions
Use low-bind siliconized tubes to minimize protein sticking to plastic
Tips for during the IP
Treat lysates with Benzonase/DNase/RNase to eliminate nucleic acid–mediated false interactions
Crosslink antibodies to beads to prevent antibody leaching and allow you to wash stringently
Weak interaction can benefit from quick, room-temperature binding (15 min) instead of 4 °C—try it
Do a high-salt final wash to remove IgG and contaminants
Tips for after the IP
Match your elution to the next experiment: acidic or SDS for WB, peptide-based or on-bead digest for MS, gentle elution for activity assays
In Western blots, use TrueBlot secondaries to avoid IgG contamination bands
Two techniques to confirm interaction:
A reciprocal IP (pull down the other protein)
Orthogonal assays (PLA, pulldown, size-exclusion)
As promised, better science in 3 minutes or less.
See you next week,
— Wildtype One
🧬 Was this email forwarded to you? Use this link to join 400+ elite researchers getting weekly lab hacks and tips (it’s free).