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

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.