BPC-157 scratch assay: 38% closure, but pFAK blot disappeared

Check your lysis buffer. I lost pFAK Y397 when I used RIPA without fresh orthovanadate. Total FAK was fine. 38% closure at 18h seems high for serum-starved though.

Was acetic vehcile pH-matched? Low pH alone can push closure.

If vehicle was pH 7.2 and gave 12%, acid doesn't explain 38%. Fresh inhibitor cocktail changed my pFAK blots completely though.

I'd bet on a transient pFAK peak before 30 min. In my HUVEC scratch assays it's 5–10 min, gone by 30. Try 5, 10, 15 min.

The pERK 2.1x with missing pFAK is interesting. Could be GPCR-driven transactivation via EGFR, not integrin signaling. If you add a Src family inhibitor like PP2, does pERK drop? Also, 12h serum starvation may synchronize cells but it can upregulate some phosphatases. I'd run a phospho-tyrosine blot first to see if the issue is FAK-specific or global. n=4 is decent, but passage number matters. In my primary fibroblast preps, pFAK Y397 was almost invisible at 30 min when I used gelatin, yet pERK stayed up. That pushed me toward a FAK-independent migration readout.

Passage 4–6 here too. Global pTyr dropped at 30 min.

Wait, you saw pFAK invisible on gelatin too? I'm on gelatin now and my 30-min blot is blank. Did fibronectin fix it?

Fibronectin gave me a clean 10-min pFAK peak, but baseline closure jumped. If you switch, keep vehicle identical or you'll chase another variable.

I chased a vanishing pFAK Y397 in a fibroblast scratch assay too, and it turned out my “30 min” was really 38 min because I was lysing wells one by one; at 10 min it was 3.2x vehicle, at 20 min 1.1x, and by 30 min 0.4x, so the signal didn’t vanish, it just left before my blot. I’d bet you missed a transient peak rather than FAK-independent migration, especially with pERK still up 2.1x at 30 min. One thing that bit me: old acetic acid vehicle stock, even when final pH read 7.2, gave scruffy phospho blots across the plate, so I now make it fresh and match vehicle molarity exactly. If you can, run a same-plate mini time course at 5/10/20/30 min with orthovanadate in the lysis and check whether the 18h closure tracks the early pFAK peak or the pERK arm.

Not sure about your lysis setup, but I’d check whether you’re lysing whole wells or just the scratch zone. I remember a 2023 thread where someone’s pFAK “disappeared” because the migrating front was a tiny fraction of the well; edge-only lysates brought Y397 back. If you’re scraping the whole well, that dilution can make a real signal look gone while pERK still reads up. Also, is your Na3VO4 fresh? I had a batch go off and pFAK vanished while total FAK looked fine. What’s your lysis timing relative to the scratch—same plate or separate?

Did you lyse directly in the dish or scrape and pellet first? That’s where my pFAK Y397 kept vanishing—one extra spin and it was gone. Also run a no-scratch, serum-starved control. If pFAK is flat there too, it’s your starvation/lysis, not the migration. What are you normalizing to—total protein or cell number?

I’m just a dorm rat whose cheap-protein rotation is lentils, canned tuna, and regret, so take this with salt. If total FAK is stable but Y397 vanishes, I’d check the pFAK/total FAK ratio at 5 and 15 min, plus whether your phosphatase inhibitors were fresh. pERK up 2.1x could mean ERK is driving migration while FAK Y397 gets turned over rather than never activated. What’s your pFAK/total ratio, not just blot intensity?

pERK 2.1x with pFAK MIA doesn’t scream FAK-independent to me yet. What’s your pFAK positive control? I’d toss in a pervanadate-treated lysate—if Y397 comes back, you’re losing phosphate during collection, not biology. Also, Y397 is low-abundance; total FAK can look stable while pFAK drops below detection if you’re loading near the floor. What’s your µg/lane and antibody dilution?

Nice n. One thing that’s bitten people in past scratch threads: when you add the peptide relative to the scratch. Pre-incubating before wounding gave a clean pFAK peak in an old EGF thread, while adding it right at t=0 after the scratch wash sometimes blunted Y397 even though closure still happened. Also, are you snap-freezing immediately or imaging first? A 3–4 min RT delay tanked my pFAK once. If pERK is up and closure is reproducible, I’d maybe check paxillin/vinculin at the leading edge by IF instead of fighting the blot. What’s your exact add-vs-scratch order?

Tbh That pFAK vanishing act would drive me nuts. Did you add apyrase to the scratch medium? Wounding dumps ATP from damaged cells, which can spike pERK and transiently mess with FAK signaling, so vehicle+apyrase might reveal the real BPC-157 effect. I’d also run a FAK inhibitor arm and, if clsure still happens, call it FAK-independent motility/proliferation. One more: are you serum-starving in the same plate you scratch? My old lab saw medium change itself shift pFAK. Keep at it — I’m in the regain-and-restart club, and stubborn experiments feel weirdly similar.

Six weeks and three passages in, I’d also wonder if the fibroblasts are drifting. In an old scratch-assay thread here, the pFAK Y397 disappearing was partly lysis speed: plates sat on ice while people counted, and phosphatases won. I now scrape directly into fresh PI/vanadate and freeze immediately; made a bigger difference than swapping substrates. If pERK is still up at 30 min, I’d run a tight 5/10/15/30/60 time course and include a no-scratch control on the same plate. Was your 30-min lysate frozen once or re-probed from an earlier blot? That one burned me before.

Not BPC, but I ran scratch assays on primary fibroblasts and pFAK Y397 was the flakiest readout. My culprit was lysis: scraping in plain cold PBS without fresh phosphatase inhibitors made pFAK vanish while pERK looked great. Now I add inhibitors and freeze on dry ice immediately. Did you run an unscratched confluent control at 30 min? If pFAK is low there too, it’s probably your window/line, not the peptide. And are you normalizing pFAK to total FAK per lane or to GAPDH? That flipped my whole story once.

ngl not a lab wizard but i track assay stuff obsessively. did you run a no-scratch + peptide control? pFAK Y397 can tank just from low density/wound edge, not the peptide. also 30 min might be late; i’d spot-check 5/10/15 min and 2h. if pERK is up, could be ERK-driven motility that doesn’t need FAK. what’s paxillin Y118 doing? that’d tell you if adhesions are

not a BPC expert, just a dorm-microwave scientist. Did you include an unscratched + peptide control at 30 min? In my hands scratch alone can yank pFAK Y397 around, so without that control it’s hard to separate “peptide killed FAK” from “scratch + peptide changed focal adhesion turnover.” Also, if pERK is up, I’d blot paxillin Y118 or pFAK Y576/577 before writing off FAK. Cheaper than repeating n=4, and it’ll tell you if it’s just that one phospho-site being flaky.

I chased this exact ghost in my own scratch runs: Y397 pFAK peaked at 8 min (3.4x), then dropped below vehicle by 20–30 min. My paxillin Y118 stayed 1.8x at 30 min. So before calling it FAK-independent, I’d plot 0/5/10/15/20/30 and probe pY861 or paxillin. Also, Y397 is autophosphorylation and very phosphatase-sensitive; if the lysate wasn’t snap-frozen with inhibitors, it can vanish while total FAK and pERK look fine. Did your lysis protocol include a phosphatase inhibitor cocktail?

ngl this reads like a whole-well vs wound-edge problem. a 30 min pFAK blot can look vanished if the signal is a tight rim at the leading edge and you lysed the entire well; the migrating front is maybe 10–20% of the area. i’d fix a time series (10/20/30/60 min) and do pFAK Y397 IF at the scratch edge, then quantify per wound area, not per well. also consider a phosphatase-inhibitor cocktail in lysis or a pervanadate pulse. did you normalize pFAK to total FAK in the same lane or total protein? and was the 30 min lysis right after the serum-free/peptide media swap? that swap can spike/de-spike pFAK fast.

Two things I’d check before calling it FAK-independent: fresh vanadate/cocktail in the lysis buffer and whether you’re losing the pFAK pool to the Triton-insoluble fraction. I had a similar vanishing pFAK once—total FAK fine—because lystes sat on ice too long before boiling. Are you snap-freezing immediately? Also, fibronectin vs plain plastic can swing baseline Y397 a lot. If the scratch is on uncoated plastic, 38% closure plus pERK up might be more about ECM-independent motility. What coating are you using?

Data-calm take: one 30-min blot is a single weigh-in, not a trend. The 38% closure + pERK up feels like real signal; pFAK Y397 can be stupidly transient and dephosphorylates fast if cells sit on ice. Concrete q: did you lyse straight into hot Laemmli or use a phosphatase-inhibitor cocktail with immediate snap-freeze? Also, did you run a 5/15/30/60 min time course with vehicle at each point? Scratch alone can spike pFAK in controls, so the “disappearance” may just be your peptide arm falling below an already-elevated vehicle baseline. If the functional readout holds across passages, I’d trust the trend line over one frame.

Night shifter here. Check earlier timepoints. pFAK Y397 is often biphasic: 10–15 min spike, then gone by 30. If you only blotted 30, you missed it. Also add a FAK inhibitor to the scratch. If closure still hits 38%, it’s FAK-independent. Then check paxillin

Did you lyse the whole well or just the scratch front? pFAK Y397 is hugely mechanosensitive and often restricted to the leading edge; whole-well lysates can dilute it to noise even when migration is FAK-dependent. I’d try IF for pFAK at the edge or a quick leading-edge scrape/lysate, plus keep lysates ice-cold with fresh phosphatase inhibitors. Also, any chance the 30 min timepoint is past a very early pulse? In my hands pFAK can spike and fall within minutes. What’s your lysis timing from plate to ice?

Not a BPC guru, but I’ve lost pFAK Y397 before: my lysis buffer was missing vanadate/fluoride and tubes sat on ice while I hunted for a marker. Total FAK fine, pFAK nuked. Now I snap-freeze with phosphatase inhibitor cocktail. Did you blot Y861 or Y925? If only Y397 vanishes, could be site-specific phosphatase activity rather than FAK-independent migration. What’s your lysis recipe? (My dorm fridge is 90% energy drinks, so I respect the 6-week grind.)

Not a BPC person, but our lab had this exact “pFAK ghost” when we lysed whole wells. The leading-edge cells are a tiny fraction, so Y397 can vanish in bulk lysate even if the edge is hot. Did you scrape just the migrated front or do IHC on the scratch? Also, 38% closure at 18h could be proliferation creeping in; a quick EdU or mitomycin-c pre-treat would separate migration from growth. If pERK is up, that’s my first suspect. Keep us posted—this is the kind of assay that makes you want to call your insurance just to hear a calm voice.

One thing I’d check is whether your lysis buffer actually held phosphatase inhibition. I once lost pFAK Y397 completely by 30 min because my vanadate was old; fresh Na3VO4/NaF plus snap-freezing brought back a clear 5-min peak. If you can, run a tiny time course at 2/5/10/15/30 and also blot paxillin Y118. If that’s flat too while migration still happens, I’d lean ERK/other-path. Were the pERK and pFAK blots from the exact same lysate prep, or different days?

Airport-gate scientist here: I’d stop thinking whole-well lysate and check the wound edge specifically. If your 30-min blot is from the whole well, the migrating rim can be a tiny fraction of total protein, so pFAK Y397 can look “gone” while total FAK stays stable. Try a narrow scrape across the closed strip, or pFAK immunofluorescence at the leading edge. Also, lysis without fresh vanadate/phosphatase cocktail can eat Y397 fast. Did you see any pFAK at 5–15 min, or is 30 min your earliest point?

Hi, total newbie here, so ignore if obvious—was the pFAK blot harvested 30 min after peptide addition, or 30 min after the scratch itself? If the scratch causes an early FAK spike that resolves by 30 min, your 38% closure could be downstream/parallel. Did you also run a lysis control with fresh phosphatase/EDTA-free inhibitors? I’ve seen pFAK Y397 disappear while total FAK stays stable when that step is off. Sorry, small nervous question—just curious!

Honestly, if total FAK is stable, I’d stop chasing Y397 for a sec and blot paxillin Y118 or p130Cas, plus maybe pFAK Y576/577. In the trials, Y397 can be a lousy proxy for active FAK at one timepoint—Src-dependent sites and downstream substrates often tell a cleaner story. Also, are you lysing the whole well or just the migrated edge? Whole-well lysate can dilute leading-edge pFAK below detection while pERK from bulk cells still looks up. I wasted weeks on that. Did you image the scratch before lysis to normalize closure per well?

Did you run a no-scratch serum-starved control next to those 30-min lysates? Scratch itself can spike pFAK Y397 in vehicle, so BPC-157 might be blunting the injury response rather than FAK-independent migration. Whole-well blotting also dilutes leading-edge signal. If you can, do pFAK IF and compare wound front vs trailing cells—if it’s there but absent in lysate, it’s localization/dilution, not biology. Also, were lysates flash-frozen fast? Y397 is brutally phosphatase-sensitive.

hi, week-one newbie here, so please forgive a naive question. Did you run a general phospho-tyrosine blot, or only pFAK Y397? If global pTyr is fine but Y397 is gone, that points more to site-specific dephosphorylation than total FAK loss.

Not a BPC wizard, just a small-town pharmacy counter lurker who tracks weird assay ghosts. Did you lyse the whole well or just the scratch edge? In my scratch runs, pFAK Y397 was really edge-enriched, so whole-well lysis diluted it below blot detection even when migration was real. Also, are you adding fresh orthovanadate/phosphatase inhibitor to the lysis buffer? I lost a pFAK signal once because my inhibitor was old—total FAK looked fine but phospho vanished. If you can, run a quick pY20 or pFAK on a concentrated edge scrape from the same lysate. Insurance wouldn’t cover my curiosity, but it saved me a repeat.

Not a BPC person, but I’ve burned months on scratch/WB timing. If 30 min is your only point, you likely missed it. Y397 peaks ~5–15 min post-injury, then can drop below baseline via dephosphorylation/trafficking. Run a mini time course: 0, 5, 15, 30, 60. Also, did you lyse whole well or only wound edge? Edge vs bulk pFAK can move in opposite directions.

Oof, pFAK vanishing while pERK climbs is such a mind-bender. Was your 30 min after peptide addition or after wounding? In my primary fibro scratches, wounding alone can sike pFAK early and then it drops hard by 30 min, especially with a 12h serum starve. Also, same-day vs freeze-thaw lysates? I’ve lost Y397 after one freeze-thaw while total FAK looked fine. If you still have lysate, paxillin Y118 or p130Cas might tell you whether it’s FAK-independent adhesion turnover.

not a BPC person, but I run a cafe and live by timing windows—espresso goes from sweet to ash in seconds, so I’d ask: was your 30 min lysate collected after peptide addition or after the scratch? pFAK can be a 5–15 min spike and then get dephosphorylated; by 30 min pERK can still be up while pFAK is already back down. Also, whole-well lysates dilute the leading-edge cells. If you can, scrape just the migrated front or try a 5/10/15/30 min series with phosphatase inhibitors in the lysis buffer. Did your total FAK stay stable across all those time points?

Not BPC-specific, but Y397 is a phosphatase magnet. Did you add fresh Na3VO4/okadaic acid to lysis buffer and keep everything ice-cold? I lost pFAK once just from a 10-min bench delay. Also, a 30-min single point won’t rule out a transient 5–15 min spike. Try

Hi, I’m brand new here (week one), so sorry if this is obvious. Could it partly be a sampling problem? If you lysed the whole well, wound-edge cells are a small fraction, so pFAK Y397 could just be diluted while total FAK stays stable. Have you tried scraping only the leading edge, or pFAK immunofluorescence at the wound border? Also, were all three passages lysed at the exact same 30 min post-scratch, same cell density? I track everything obsessively in my own life and still miss batch timing. Ignore if dumb.

Not a BPC expert either, but the whole-well vs leading-edge thing bit me. If you’re blotting the entire well, a 38% closure can dilute pFAK signal from the migrating front. I started lysing just the scratch corridor after a quick PBS wash, plus fresh orthovanadate and no EDTA in the RIPA, and Y397 became detectable. What’s your lysis buffer/timing? Also, are you normalizing pFAK to total FAK in the same lane or to protein from the edge? That can flip the story.

ngl quick dumb question from the cheap seats: did you run a proliferation control at 18h? I’ve had scratch assays look like migration when it was really just cells doubling into the gap—especially with pERK up 2.1x. Serum starvation helps but doesn’t always stop it. EdU or just counting nuclei in the gap would separate migration from proliferation

Hi, super new here so sorry if this is obvious—did you sample earlier than 30 min? In scratch assays

Not a FAK person, but whole-well lysates have burned me in scratch assays: at 30 min the migrating edge is maybe 5% of the dish, so active pFAK gets averaged into nothing while bulk FAK stays flat. Did you dissect/lyse just the wound strip, or is that blot from the whole well? If it’s full-well, I’d trust the pERK before the pFAK dropout.

Been there. If total FAK is rock-steady but pY397 vanishes in whole lysate, I’d check whether you’re scraping straight into RIPA or letting plates sit. I got wildly different pFAK when lysis was slow—phosphatases chew it fast, and ERK can still be up. Also, with only 38% closure, the actual leading edge is a tiny fraction of the well. Whole-lysate Western may miss a focal-adhesion pool that paxillin/pFAK IF would catch. Did you normalize pFAK to total FAK per lane, or just compare band intensity? That detail burned me on a similar scratch assay.

One thing I’d check before calling it FAK-independent: what does vehicle pFAK look like at 30 min? In my scratch assays, wounding alone spikes pFAK around 5–10 min and it can fall below baseline by 30, so a peptide effect could just shift that curve earlier. If vehicle is still strong at 30 and only treated is gone, that’s more interesting. Also, were lysates snap-frozen right away? I’ve had Y397 vanish from even brief bench delay unless phosphatase inhibitors were fresh.

I do scratch assays in 10-minute chunks between school pickup, so grain of salt. Did you normalize closure to initial wound area per well, or just one width measurement? Uneven edges once faked a 30% difference for me. Also, if pFAK is confined to leading-edge cells, whole-well lysate can dilute it to background by 30 min—did you try IF or a little edge scrape? And did you throw in mitomycin C to rule out proliferation? My snack drawer is labeled; my plate maps are chaos.

Wait, are you lysing the whole well or just the wound? If pFAK is only in the leading-edge cells, bulk lysate from all the quiescent fibroblasts can dilute it into nonexistence—like my dorm “high-protein” chili, where one can of beans disappears into a vat of rice. Try wound-only lysis or pFAK IF on the same coverslip, then normalize to edge length. Also, how confluent were the outer cells? That paracrine mix can matter more than people admit.

yo, different angle: are you sure that 38% is migration and not cells dividing into the gap? pERK up + pFAK gone makes me suspicious. Did you run a mitomycin-C or low-serum/serum-free control arm? Also check if your image analysis is registered to the same ROI—wound-edge cells lifting/detaching can look like closure. Quick EdU or nuclei count across the scratch would tell you if proliferation is doing the work. If it is, the FAK blot might be a red herring. Not a lab wizard, but that’s the first control I’d add.