5:2 fasting paper replication: BDNF flat but p-TrkB up?

Mature vs proBDNF first. Many ELISAs cross-react, so total BDNF can look flat even when signaling changes.

We saw total BDNF flat btu p-TrkB up 1.8x. Snap-freeze time mattered more than diet, teh variance was huge.

So mature vs proBDNF - did you run the same antibody clone as the paper?

I'd be careful blaming the assay before checking stress. In our 5:2 mouse cohort, corticosterone at sacrifice explained ~25% of BDNF variance; singly housed mice looked 'non-responders' even when p-CREB was up.

Did the original paper control for cage changes and sacrifice time? If they used CO2 vs decap, that alone can shift phospho signals.

Your Y816 data may actually be the cleaner endpoint.

What does p-TrkB look like in the non-fasting controls across the day? Also, were the ELISAs run on whole hippocampus or microdissected dentate? Regional differences could easily flatten a 38% whole-hippo effect.

Ventral dentate only for us. Dorsal barely moved.

That matches our IHC. p-CREB bright in ventral dentate, dorsal flat. Are you cryosectioning fresh or fixed?

If p-TrkB and p-CREB agree, I'd trust the mechanism over total BDNF. Maybe the paper's 38% was timing luck.

Six weeks in and your p-TrkB is 2.1x while total BDNF is only +12%? I’d flip your worry: that smells less like a missed processing step and more like the original ELISA was catching a transient peak your week-8 timepoint is sitting downstream of, because p-CREB +45% doesn’t come from nothing.

My own n=1 5:2 run back in Feb–Apr (two 500-ish kcal days, same Mon/Thu slots) had the same lag shape—fasting glucose shifted by week 3, waist didn’t budge until week 9, and the scale only moved after I started weighing Fridays and stopped daily checks, which had me eating back ~300 kcal on fast days. For your replication, did you counterbalance sacrifice order across fasted/fed groups and run a naive-fed plate control?

I once got a gorgeous fake p-TrkB signal because the fasted cage got pulled first and sat in the procedure room 6 extra minutes. If your controls are clean, I’d trust the phospho data over the BDNF ELISA and maybe add a proBDNF/BDNF ratio blot before blaming your protocol.

Before you touch another ELISA, check whether week 8 landed on a fasting day or a refeed morning—I did a 10-week 5:2 human run and my scale/waist looked like a failed replicate until I realized I was measuring after different weekend refeeds; same 7am time, 2.1 lb and 0.75 inch spread, which was enough to make me stop trusting a single timepoint. If your week-8 pull was after a refeed while the original was at the tail of a fast, that could explain a chunk of the gap, and it’s a 10-minute protocol note to chek before chasing antibodies.

not a lab rat but i'd check whether your BDNF ELISA and p-TrkB came from the same lysate/hemisphere. if separate preps, freeze-thaw or homogenization time can tank mature BDNF while phospho-epitopes hang on. also did you run proBDNF or just mature?

a cleavage/processing shift could give flat total BDNF but more TrkB signaling. i track my own fast/refeed by sleep and mood and it's nonlinear af, so week 8 variance is relatable.

Honestly, Small-town pharmacy brain here, so grain of salt. Could your BDNF ELISA be reading proBDNF too? If total BDNF is flat but mature BDNF is up, p-TrkB Y816

not a bench wizard, but: are you normalizing p-TrkB to total TrkB, not just GAPDH? If total TrkB slipped, that 2.1x could be partly a denominator artifact. Also, how long from dissecton to freezing?

Phospho-epitopes can tank fast, and a 5:2 schedule might change how much you’re rushing. I’m a dorm-food guy—my protein tracking is just tuna packets and Greek yogurt cups—but even I know cold-chain timing wrecks everything. Did you run phospho/total on the same membrane?

Did the original methods specify whether their BDNF ELISA detected proBDNF, mature BDNF, or both? That’s my first suspect.

p-TrkB Y816 is mainly a mature-BDNF readout, so if 5:2 shifted cleavage (tPA/plasmin, maybe MMP-9) rather than total synthesis, you could get flat total BDNF by ELISA while p-TrkB and p-CREB climb.

I’d also check dissection/ischemia time, since proBDNF can degrade or leak fast. If you have leftover lysate, a mature-BDNF Western or proBDNF antibody could show whether the ratio moved.

What did their methods say about extraction time and antibody specificity?

Honestly, One thing I’d check: were fasted vs control mice in the same cage/enrichment setup? In my own habit tracking, environment (sleep, steps, stress) often moved my mood/inflammatory markers more than the fasting window itself.

For mice, social housing, bedding changes, or running-wheel access can shift hippocampal BDNF enough to blur a 38% effect. Also, did you verify the 5:2 days by weighing food intake, not just assuming they ate nothing? If controls hoarded chow, the contrast shrinks.

Rooting for your p-TrkB/CREB signal—sounds like the receptor side is responding even if ELISA isn’t.

Mechanism nerd, not a bench wizard: total tissue BDNF ELISA mostly reflects intracellular pools, while p-TrkB

Spreadsheet nerd here. Did your BDNF ELISA detect total BDNF (pro + mature) or mature only? Most standard kits are total, so +12% total could still hide a big shift from proBDNF to mature BDNF, which would fit p-TrkB Y816 up without a huge ELISA delta.

I’d run western for proBDNF vs mature and add p-ERK1/2/p-Akt to see if the canonical TrkB arm tracks. Also check perfusion: residual platelet BDNF from non-perfused hippocampus can flatten your ELISA. What’s your CV per group?

With n=12/group, +12% at p=0.21 is almost exactly underpowered—not necessarily a replication failure.

Cafe owner here, so portion control is my daily boss fight, but I do a 5:2-ish rhythm around pastry trays. The thing that bites me is timing relative to the fast, not clock time. Did you match fast day vs refeed day, not just Zeitgeber time?

My energy/mood tanks on fast mornings but rebounds after the first meal. If the original samples were collected after an overnight fast and yours after a refeed day, BDNF could look flat even while TrkB signaling is up.

I’d split proBDNF from mature BDNF before blaming the protocol. Most ELISAs don’t, so +12% total can hide a real cleavage shift. p-TrkB Y816 up with flat BDNF also smells like reduced dephosphorylation—PTP1B/STEP—not more ligand. How long from decap to freeze?

Hippocampal BDNF tanks fast; phospho-epitopes hang around longer, which would give exactly your mismatch. And if both sexes are in there, estrous cycle is extra variance.

Not a vet, but I ran a 5:2-ish block for a year and tracked way too much of it. Question: was your week-8 harvest on a fasting day or after refeed? Hippocampal BDNF swings with refeeding, so a 24h offset alone can flip direction and turn your +12% into noise.

If the original killed fasted and you killed fed, then p-TrkB/p-CREB is your real signal — signaling up while ligand sits flat usually means sensitivity shifted, not supply. Did you run a fasted-vs-refed arm, or is week 8 your only timepoint?