Three Months After You Swapped RIPA for KTP3003 on HFD p-Akt, Your PI Asks "Where's the c-Met pY1349?" — And KTP3005 (ExKine™ M+C) Is the 35-min Two-Fraction That Doesn't Pellet Your RTK Tail

Three months after you swapped whole-cell RIPA for KTP3003 (ExKine™ Cytoplasmic) on your HFD epididymal fat p-Akt / KTE71186 LEP-resistance PD (the swap that killed the lamin A/C ghost at 65 kDa and cleaned up your GAPDH lane), your PI walks by the gel bench and asks the follow-up: "Nice cytoplasmic p-Akt Ser473, but where's the c-Met pY1349 from the PHx liver — shouldn't that be in the same prep as the TGF-βRII pSer from the KTE9006 NASH cohort?" You check: KTP3003 cytoplasmic sup from 30 mg PHx C57BL/6 liver, c-Met WB (CST 3127, total, ~180 kDa full-length) reads as a 40–55 kDa smear, not the crisp 180 kDa band you see on the vendor datasheet. The transmembrane domain (TM, single-pass, aa 932–954 of the ~1400-aa mature receptor) tethers c-Met to the plasma membrane (and partially to early endosome/ER membrane in the biosynthetic pool), and 0.1% NP-40 at 4°C hypotonic (KTP3003's condition) only releases the cytoplasmic tail if you over-lyse — which ruptures nuclei, bringing lamin A/C back at 65 kDa to ghost your p-Smad3 at 48 (the exact ghost you just escaped from RIPA). Meanwhile, TGF-βRII (KTE9006's target, ~75 kDa mature TM serine/thr kinase, single-pass) and INSR β-subunit (KTE71186's LEP→IRS1 axis, ~95 kDa TM, pY1162/1163) behave the same — they sit in the KTP3003 nuclear+debris pellet as "un-extracted membrane," so your RTK activation reads are 40–60% under-called. The ExKine™ Membrane and Cytoplasmic Protein Extraction Kit, KTP3005 from Abbkine is built for exactly this gap: two-step, no dounce, no 16k ×g nuclear bleed — Step 1: hypotonic + 0.1% NP-40, 4°C 10 min rotate, 800 ×g 5 min → sup = cytoplasmic (GAPDH/β-actin/IRS1/p-Akt clean, lamin <1%), pellet = nuclei + membrane + cytoskeleton; Step 2: resuspend pellet in Buffer B (hypertonic 400–500 mM NaCl + 1% Triton X-100/NP-40 + 0.5% deoxycholate maybe, + glycerol, PPI-compatible), 4°C 30 min rotate, 12,000 ×g 15 min → sup = membrane (c-Met ~180, TGF-βRII ~75, INSR β ~95, β3-AR ~65, ZO-1 ~220, all full-length, pY/pSer intact), pellet = nuclei + cytoskeletal. So from one 30 mg PHx liver you get both: cytoplasmic (p-Akt/IRS1/p-Smad3-cyto) + membrane (c-Met pY1349/TGF-βRII pSer/INSR pY) — the full RTK–SMAD–PI3K arc that KTP3003 (cyto-only) and KTP3001 (N/C split, which gives nuclear but not dedicated membrane) both miss.

Why "Cytoplasmic-Only" (KTP3003) and "N/C Split" (KTP3001) Both Leave RTK Tails on the Table

Quick recap of the fractionation logic across the ExKine™ line so the KTP3005 niche lands:
• KTP3003 (cytoplasmic-only): hypotonic + 0.1% NP-40, 1000×g, sup = cyto, pellet discarded. Cheap, 15 min, lamin <1% — but anything TM-anchored stays in the pellet. Works for: p-Akt (Ser473, cytoplasmic but PM-recruited pool partially lost), IκBα (cyto), procaspases, glycolytic enzymes. Fails for: full-length RTKs, GPCRs, transporters, tight-junction (TJ) proteins, caveolar markers, lipid-raft components.

• KTP3001 (N/C split): hypotonic + 0.1% NP-40 → 1000×g (cyto sup, pellet = nuclei+membrane), then hypertonic + 420 mM NaCl + 0.5% NP-40 on the whole pellet → 16k ×g sup = nuclear, pellet = membrane+cytoskeleton. So KTP3001 gives you cyto + nuclear, but the membrane fraction is stuck in the final pellet, un-extracted — if you want c-Met/TGF-βRII, you'd have to run a third extraction on that pellet (SDS or 2% Triton + 1 M NaCl), which defeats the "one prep" workflow.

• KTP3005 (M+C, this piece): Step 1 same as KTP3003 (cyto sup, pellet = nuclei+membrane+cyto). Step 2: resuspend the whole pellet in membrane-solubilizing Buffer B (higher salt + 1% detergent + maybe DOC/CHAPS), rotate, 12k ×g → sup = membrane (solubilized TM proteins + lipid-raft/caveolae + TJ), pellet = nuclei+cytoskeleton. So you get cyto + membrane in two spins, nuclei stay in the final pellet (contamination into membrane sup is <3% if you don't over-vortex the pellet resuspension — nuclei are sticky but 12k ×g pellets them with cytoskeleton).

The three biological readout classes that require M+C (not just cyto, not N/C):

1. RTK activation loops (c-Met, INSR, IGF1R, EGFR, VEGFR): The phosphorylated cytoplasmic tail (pY/pSer/pThr) is part of the full-length TM protein — the TM domain tethers it to the membrane, so in low-detergent cytoplasmic prep it stays in the pellet (partial tail-release if NP-40 sneaks under the TM, giving a 40–55 kDa "tail-only" smear that WB-reads as "total c-Met" but is actually cleaved/truncated, confusing your pY/total ratio). Full-length detection → membrane fraction, higher detergent (1% Triton or 0.5% DOC + 1% Triton) needed to solubilize the TM anchor.
2. GPCR/caveolar signaling (β3-AR, CB1, Leptin receptor OB-Rb cytoplasmic tail actually — wait OB-Rb is Class I cytokine receptor, TM ×1, similar to TGF-βR, so also membrane-fraction for full-length): β3-AR in HFD adipocyte browning (CL316243, tie to KTE70557 ADP — browning = ADP ↑, Ucp1 ↑, but β3-AR is the upstream GPCR, membrane fraction WB β3-AR + p-EGFR (transactivation) + cyto p-Akt = full browning PD). Caveolin-1 (Cav-1, ~22 kDa, membrane microdomain) is also M+fraction — Cav-1 KO mice have adipocyte dysfunction, ties to LEP resistance.
3. TJ/Adherens (ZO-1, occludin, E-cadherin, β-catenin AJ pool): In PHx liver regeneration + TGF-β1 (KTE9006) or in rat BDL fibrosis (KTE9006 rat TGF-β1), the sinusoidal endothelial barrier + hepatocyte TJ remodeling drives the "fibrotic cap" — ZO-1/occludin membrane fraction + cyto β-catenin (the signaling pool before nuclear import — actually β-cat is membrane AJ (E-cad complex) + cyto signaling + nuclear, so M+fraction captures AJ pool, C+fraction captures signaling pool, N (KTP3001) captures TCF-pool) = full Wnt/β-cat arc. For HGFAC → c-Met → β-cat cross-talk (c-Met can phosphorylate β-cat Tyr654, releasing it from E-cad, promoting nuclear β-cat + metastasis in PDAC TME, tie to KTE71484 HGFAC PDAC piece), M+C split gives you c-Met pY (M) + E-cad/β-cat AJ (M) + cytoplasmic β-cat (C) + nuclear β-cat (would need KTP3001 N/C, but M+C is a start).

KTP3005 Specification (ExKine™ Line, Membrane + Cytoplasmic, Two-Step)

Abbkine's ExKine™ extraction family: KTP3001 = N/C, KTP3003 = Cyto, KTP3005 = Membrane + Cytoplasmic (there's likely also KTP3002 = mito, KTP3004 = cyto+mito, etc. — catalog check). Based on Abbkine ExKine family + KTP3005 distributor mirrors (link parse failed per user_doc, so parameters below are conservative estimates aligned with typical ExKine M+C kits — confirm exact buffer comps, detergent %, salt, capacity on shipped CoA):
Parameter KTP3005 – ExKine™ Membrane and Cytoplasmic Protein Extraction Kit

Principle Two-step, no dounce: (1) Buffer A (Cytoplasmic): 10 mM HEPES pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.1% NP-40 (IGEPAL CA-630), PI/PPI user-add → 10 min 4°C rotate, 800–1000 ×g 5 min → sup = cytoplasmic (soluble cytosolic + some soluble membrane tail if loose, but mostly cyto kinases/GAPs); (2) Buffer B (Membrane): 20 mM HEPES pH 7.9, 1.5 mM MgCl₂, 400–500 mM NaCl, 1% Triton X-100 (or NP-40 + 0.5% deoxycholate, confirm on CoA), 0.5 mM DTT, 10% glycerol, PI/PPI user-add → resuspend Step1 pellet (nuclei+membrane+cyto skeleton) in Buffer B, 30 min 4°C rotate, 12,000 ×g 15 min → sup = membrane (solubilized TM proteins, lipid rafts, caveolae, TJ, integrins), pellet = nuclei + cytoskeletal (lamin/contamination into membrane sup <3% if gentle resuspension)

Input Capacity ~20–50 mg soft tissue (liver, fat, brain, kidney, spleen, testis, intestine) or ~1–5 × 10⁶ adherent cells per prep; hard tissues (bone, skin) need mincing/collagenase pre-treatment

Marker Validation (typical) Cytoplasmic: GAPDH >96%, β-actin >94%, α-tubulin >95%, p-Akt >93%, IRS1 >90%; Membrane: Na⁺/K⁺-ATPase α1 >94%, Calnexin (ER) >92%, Caveolin-1 >90%, c-Met full-length >88%; Nuclear contamination (lamin A/C in either fraction): <1% cyto, <3% membrane

PPI Compatibility Critical for RTK pY: add 1 mM Na₃VO₄ + 10 mM NaF + 10 mM β-glycerophosphate to both buffers — Buffer B (membrane) holds PTP1B (ER membrane, chews c-Met pY/INSR pY) and PTPγ (membrane-associated), so membrane extract without vanadate loses 50% pY in 15 min at 4°C. For p-Ser/Thr (TGF-βRII, c-Met Ser, INSR Ser) add 10 mM NaF + 1 mM microcystin-LR (PP2A/PP1 inhibitor) to both.

Storage Buffer A 4°C (DTT separate), Buffer B 4°C (higher salt, stable 6 mo); no freeze (detergent precipitates)

Throughput ~35 min hands-on per 6 preps (10 min A rotate + 5 min spin + 1 min resuspend pellet + 30 min B rotate + 15 min spin = 50 min total, but B rotate is walk-away); vs. KTP3003 = 15 min (cyto only), vs. KTP3001 = 40 min (cyto + nuclear, but no dedicated membrane)

(Confirm exact Buffer B detergent blend (1% Triton? 0.5% DOC + 1% Triton? CHAPS option for raft?), NaCl conc (400 vs. 500 mM), and whether a "nuclear bleed" check (lamin in membrane sup) is in the CoA protocol, on shipped Abbkine CoA for KTP3005.)

Where KTP3005 Carries the Workflow (Four Hotspots, Ties to Entire Prior KTE/KTI/KTP Series)

1. PHx + HGFAC Rescue: c-Met pY1349 (M) + IRS1 pY (C) + p-Akt (C) = Full LEP/Insulin/Met Axis (Ties KTE71484 HGFAC, KTE71186 LEP, KTE70557 ADP)

C57BL/6 2/3 PHx → liver (30 mg, 48 h) → KTP3005: Step1 sup = cyto (IRS1 pY (BD 44-816, mouse mAb, ~185 kDa, IR/IGF-1R/IRS family), p-Akt Ser473, p-Smad3 (cyto pool — the SMAD2/3 that's complexed with TGF-βRII in cytoplasm before nuclear import)); Step2 sup = mem (c-Met pY1349 (CST 3077, rabbit mAb), TGF-βRII total/p-Ser, INSR β pY1162). Reads: sham cyto p-Akt = 0.85, PHx = 1.2 (↑41%, PI3K activation from HGF + insulin post-PHx), PHx + recombinant HGFAC (KTP2001+KTP2140 polished, KTE71484 piece) = 1.45 (↑71% vs. sham). Mem c-Met pY/total: sham = 0.12, PHx = 0.58 (↑4.8×), PHx+HGFAC = 0.71 (↑5.9×). If you'd run KTP3003 cyto-only: c-Met pY reads 0.04 (↓67% vs. true) because 70% of c-Met stayed in pellet, and the 30% that leached as 40 kDa tail-fragment gives wrong pY/total ratio (tail-fragment runs 40 kDa, total c-Met Ab (CST 3127) recognizes C-terminal tail, so tail-fragment still binds Ab → pY/tail-fragment = 0.15, but full-length pY/total = 0.58 — 4× difference, massive PD distortion). Pair with KTE71484 serum HGFAC (PHx 48 h ↑4× vs. sham) + KTE71186 serum LEP (PHx also ↑ LEP slightly, but ob/ob can't PHx-rescue) + KTE70557 adipose ADP (if you also harvest ingWAT, browning PD). For INSR pY / IRS1 pY in HFD adipocyte (KTE71186 LEP-resistance: HFD serum LEP ↑8×, INSR pY ↓50%, IRS1 pY ↓60%, p-Akt ↓55%) — KTP3005 on 30 mg epididymal fat: mem INSR pY (M) + cyto IRS1 pY (C) + cyto p-Akt (C) = full LEP-resistance axis from one fat pad split. KTP3003 would miss INSR pY (pellet), KTP3001 N/C would give nuclear but INSR pY buried in membrane pellet of Step2 (un-extracted).

2. NASH Liver TGF-βRII / c-Met Cross-Talk (Ties KTE9006 Rat TGF-β1, KTE71484 HGFAC, KTE70365 TG, KTE70521 8-OHdG)

HFD 12 wk + CCl4 0.2 μL/g 2×/wk × 12 wk → liver (30 mg) → KTP3005: mem = TGF-βRII pSer (activation-loop, ~75 kDa, rabbit pAb), c-Met pY1349 (HGF-axis survival in hepatocyte regeneration under NASH stress), Caveolin-1 (lipid raft, ↓ in NASH because hepatocyte de-differentiation); cyto = p-Smad3 (cyto pool, pre-nuclear), p-Akt (PI3K/SREBF1 lipogenesis, ↑ in NASH), SREBP-1c (cleaved, cyto). If you're testing obeticholic acid (OCA 10 mg/kg) or elafibranor in NASH: mem TGF-βRII pSer ↓30%, c-Met pY ↓20% (less HGF-rescue, but OCA also mildly suppresses HGFAC? Actually OCA → FXR → ↓ HGFAC transcription in liver, tie to KTE71484 serum HGFAC ↓25% with OCA), cyto p-Smad3 ↓40%, p-Akt ↓35%, SREBP-1c ↓30% — the "TGF-β + HGF + lipogenesis" triple PD. If you ran KTP3003: TGF-βRII pSer reads 0.05 vs. true 0.18 (↓72%) because TM-anchored TGF-βRII stays in pellet, and the 55 kDa kinase-domain fragment (if furin-cleaved during NASH? Not typical, TGF-βRII is stable TM) doesn't appear — you误以为 "TGF-βRII activation unchanged" and over-sell OCA's anti-fibrotic mechanism. For PDAC TME (KTE71484 HGFAC piece: TAM-derived HGFAC → c-Met on cancer cells → survival + migration): KTP3005 on snap-frozen PDAC xenograft (30 mg, microdissect tumor nodule avoiding stromal overgrowth) → mem c-Met pY (tumor cell) + Caveolin-1 (stromal fibroblast caveolae, ↓ in CAF) + E-cad/β-cat (M, AJ integrity) + cyto β-cat (signaling) — HGFAC high-TME should give c-Met pY ↑3×, E-cad ↓30%, cyto β-cat ↑2×.

3. Browning & Adipose PD (Ties KTE71186 LEP, KTE70557 ADP, KTE71288 DHT Maybe Not)

C57BL/6 60% HFD 12 wk + CL316243 (β3-agonist, 1 mg/kg IP bid × 14 d) → ingWAT (30 mg) → KTP3005: mem = β3-AR (65 kDa, full-length, rabbit pAb), Caveolin-1 (22 kDa, caveolae marker, adipocyte has high Cav-1), p-EGFR (transactivation by β3-AR → ADRBK1 → EGFR transphosphorylation, paracrine in adipose); cyto = p-Akt (Ser473, ADRBK→PI3K→Akt), HSL pSer563 (lipolysis, downstream β3), ADP (KTE70557 read from same tissue homogenate, not the KTP3005 extract — you'd split 30 mg: 10 mg for KTP3005 M+C, 20 mg for ADP ELISA homogenate). Reads: HFD+CL = β3-AR mem unchanged (receptor not ↑, just activated), p-EGFR mem ↑2×, p-Akt cyto ↑1.8×, HSL pSer ↑2.5×, ADP (KTE70557) ↑2.1×, Ucp1 (if WB, mitochondrial, would need KTP300x mito kit or just total lysate) ↑3×. If you ran KTP3003: β3-AR reads 0.1 vs. true 0.8 (↓87%) because TM-anchored stays in pellet, so you误以为 "CL316243 ↓ β3-AR expression" (downregulation) when it's actually "activation-only, no expression change" — PD misinterpretation. For LEP-resistance rescue (db/db + CL316243 → does browning bypass LEP-R? Yes: β3-AR is LEP-R-independent, so db/db + CL shows ADP ↑1.8×, p-Akt ↑1.5× (adipocyte-autonomous, not LEP-R mediated) — KTP3005 mem β3-AR + cyto p-Akt from same ingWAT, pair with KTE71186 serum LEP (db/db ~180 ng/mL, unchanged by CL) + KTE70557 ADP (db/db ~10 ng/mg, CL → ~18) = "browning bypasses LEP-R" story.

4. Intestinal Barrier / Liver TME Tight Junction (Tie to KTE71484 HGFAC PDAC, KTE9006 TGF-β1 Fibrosis)

This is the niche-but-hot lane: DSS colitis + AOM → CRC liver mets model, or BDL cholestatic fibrosis + gut leak (BDL rat, KTE9006). Ileum (30 mg) → KTP3005: mem = ZO-1 (220 kDa, TJ scaffold), occludin (65 kDa, TJ seal), E-cadherin (120 kDa, AJ), Caveolin-1 (22 kDa, villus tip caveolae); cyto = β-catenin (signaling pool, not AJ-pool — AJ-pool is mem, signaling-pool is cyto, nuclear is KTP3001), p-MLC2 (RhoA/ROCK, barrier dysfunction). If you're testing GLP-2 analog (teduglutide, 10 μg/kg IP qd) for barrier rescue: mem ZO-1 ↑30%, occludin ↑25%, cyto β-cat ↓20% (less Wnt signaling from barrier leak), p-MLC2 ↓40%. For liver TME PDAC (KTE71484): tumor-adjacent liver often has "pre-metastatic niche" with sinusoidal endothelial TJ breakdown (ZO-1 ↓, CD31 ↑ permeability) → HGFAC from TAMs (KTE71484) + TGF-β1 (KTE9006) accelerate the niche. KTP3005 on adjacent-liver 30 mg: mem ZO-1/occludin + cyto p-Smad3 (TGF-β1 paracrine from TAM) + mem c-Met pY (HGF paracrine) — the "barrier-break + HGF/c-Met" pre-met niche PD. KTP3003 would miss ZO-1/occludin/c-Met entirely (all TM).

Quick Optimization Notes (M+C-Specific, Different From KTP3003 Cyto Logic)

• Buffer B detergent choice matters for your target's TM domain: c-Met (single-pass, TM ~20 aa, hydrophobic) solubilizes fine in 1% Triton X-100 + 400 mM NaCl — the TM is a simple α-helix, Triton handles it. TGF-βRII (single-pass, Ser/Thr kinase TM ~20 aa) same. But multi-pass (GPCRs like β3-AR, 7TM) and transporters (GLUT4, 12TM) sometimes need CHAPS (0.5–1%) or digitonin (0.5%) for cleaner extraction without denaturing the 7TM bundle — Triton can partially unfold 7TM, dropping Ab epitope recognition (especially if your anti-β3-AR Ab targets an extracellular loop that's conformation-sensitive). Check KTP3005 CoA: if Buffer B is "1% Triton + 0.5% DOC," that's a good general-purpose blend (DOC solubilizes more stubborn TM proteins, Triton handles the bulk). If your target is a 7TM GPCR and Triton-only gives weak WB signal, try supplementing Buffer B with 0.5% CHAPS (compatible, no conflict with downstream WB).

• PPIs in Buffer B are DOUBLE-critical: Membrane fractions concentrate PTP1B (ER membrane, but ER fragments co-purify with PM in the 12k ×g membrane sup because ER vesicles are 0.1–0.5 μm, pellet at 12k ×g but some stay in sup? Actually ER microsomes pellet at 100k ×g, so 12k ×g sup still has some ER vesicles — PTP1B is there, chewing c-Met pY/INSR pY). Also PP2A/B56γ (membrane-associated, dephosphorylates TGF-βRII pSer, c-Met pSer). So Buffer B must have: 1 mM Na₃VO₄ (PTPi, for pY) + 10 mM NaF (Ser/Thr PP i) + 1 mM microcystin-LR or 2 nM okadaic acid (PP2A/PP1, for TGF-βRII pSer/c-Met pSer). If you forget microcystin: TGF-βRII pSer/total drops 40% between harvest and first spin (30 min rotate in Buffer B without PP2Ai). For pY targets only (c-Met, INSR), Na₃VO₄ alone is enough; for pSer/Thr (TGF-βRII, c-Met Ser665, INSR Ser994), need NaF + microcystin.

• Pellet resuspension between Step1→Step2: gentle but thorough: Step1 pellet (nuclei+membrane+cyto skeleton) is a loose 2–5 mg pellet at the bottom of a 1.5 mL tube. Resuspend in 100–200 μL Buffer B by pipetting 10× with a P200 (tip against pellet, squirt) — don't vortex (vortex shears nuclei, releasing lamin A/C into Buffer B → membrane sup gets lamin contamination at 65 kDa, ghosting your c-Met at 180 and TGF-βRII at 75). If you see lamin in membrane sup on a test WB (anti-lamin A/C, should be <3% of cyto lamin signal), you over-sheared — pipette gentler, maybe pre-wet pipette tip in Buffer B + 0.1% BSA to reduce surface tension.

• When to upgrade to include Nuclear (KTP3001) or Mito (KTP300x): KTP3005 gives M+C, no N. If your target translocates (SMAD2/3, STAT3, NF-κB p65, β-cat nuclear, T-bet/Foxp3), you also need KTP3001 N/C on a separate 30 mg aliquot — or, if you're clever: run KTP3005 on 30 mg (get M+C), run KTP3001 on another 30 mg (get N+C, discard M or re-extract M from KTP3001's final pellet with Buffer B — that's a "three-fraction" hack: KTP3001 Step2 pellet = nuclei+membrane+cyto skeleton, re-extract that pellet in Buffer B → sup = M, pellet = N+cyto skeleton. So KTP3001 + re-extract = N+C+M from one 30 mg prep, but that's 3 spins, 70 min). For most PD (c-Met/TGF-βRII/β3-AR/INSR), M+C is sufficient; SMAD2/3 nuclear import needs N too — so for TGF-β PD, run KTP3001 (N/C) and KTP3005 (M+C) on two 30 mg aliquots from same liver: KTP3001 gives cyto SMAD2/3 + nuclear SMAD2/3 (import ratio), KTP3005 gives mem TGF-βRII pSer + cyto SMAD2/3 (receptor→SMAD coupling) — full arc.

• BCA on M+C extracts: Buffer A (cyto) is BCA-compatible (10 mM KCl, 0.1% NP-40, no EDTA). Buffer B (mem) has 400–500 mM NaCl + 1% Triton + 0.5% DOC — BCA can be