Quick answer
How do you reconstitute the KLOW stack? KLOW is a four-peptide lyophilized research blend, typically 80 mg per vial: 50 mg GHK-Cu plus 10 mg each of KPV, BPC-157 and TB-500. Warm the sealed vial to room temperature, add bacteriostatic water slowly down the glass, and swirl gently until the blue solution is clear. Adding 4 mL gives 20 mg/mL total peptide: 12.5 mg/mL GHK-Cu and 2.5 mg/mL of each of the other three. Refrigerate at 2–8 °C, protect from light, and use within about 28 days.
A four-component blend raises questions that a single-peptide vial does not: which concentration do you record, why is the liquid blue, and does one peptide limit how long the rest can be stored? This guide explains what the KLOW stack is as a laboratory blend, then walks through diluent choice, volume selection, the bench protocol, storage, and serial dilution for in vitro work. For laboratory research use only; not for human consumption.
What the KLOW stack is
"KLOW" is a trade shorthand for a co-lyophilized combination of four synthetic peptides in one vial. It extends the three-peptide GLOW blend (GHK-Cu, BPC-157 and TB-500) by adding the tripeptide KPV. Researchers use a pre-blended vial when a protocol calls for all four compounds at a fixed ratio and a single reconstitution step is more practical than preparing four separate stocks. The KLOW stack is supplied as an 80 mg vial; always confirm the per-component masses against the vial label and the batch certificate of analysis before calculating.
| Component | What it is | Approx. molecular weight | Mass in an 80 mg vial | Research context |
|---|---|---|---|---|
| GHK-Cu | Glycyl-histidyl-lysine tripeptide complexed with copper(II) | ≈ 340 g/mol (peptide); ≈ 404 g/mol (copper complex) | 50 mg | Copper-binding and extracellular-matrix gene-expression studies |
| KPV | Lysine-proline-valine, the C-terminal tripeptide of α-MSH | ≈ 342 g/mol | 10 mg | Melanocortin-related and inflammatory-signaling models |
| BPC-157 | 15-amino-acid synthetic pentadecapeptide | ≈ 1,419.5 g/mol | 10 mg | Angiogenic and cytoprotective signaling models |
| TB-500 | Synthetic thymosin beta-4 sequence (43 amino acids) | ≈ 4,963 g/mol | 10 mg | Actin-sequestration and cell-migration studies |
Two points about this table matter at the bench. First, the blend is weighted heavily toward GHK-Cu, so the "total" concentration and the concentration of any single minor component differ by a factor of eight. Second, the name TB-500 is occasionally applied by vendors to a short thymosin beta-4 fragment rather than the full sequence, so check the identity data on the COA. More background is in the KLOW stack research overview and chemical structure articles.
Why bacteriostatic water is the standard diluent
An 80 mg vial is rarely consumed in one session. Once a stopper has been punctured, every later entry is an opportunity for microbial contamination, and peptide solutions are a perfectly good growth medium. Bacteriostatic water is sterile water containing 0.9% benzyl alcohol, a preservative that inhibits the growth of bacteria introduced during repeated entries. Its performance in that role is what antimicrobial effectiveness testing (USP <51>) evaluates, and it underlies the 28-day beyond-use convention for preserved multi-use containers in USP <797>.
| Diluent | Use with KLOW? | Reason |
|---|---|---|
| Bacteriostatic water | Yes — default for multi-use vials | Preserved; all four peptides are freely water-soluble |
| Sterile water | Yes — single session or immediate aliquoting only | No preservative |
| Phosphate-buffered saline as the stock diluent | Avoid | Concentrated copper can form insoluble phosphate salts; use buffers only for final dilution |
| Anything containing EDTA or other chelators | Avoid | Chelators strip copper from GHK |
| Reducing agents (ascorbate, DTT) | Avoid in stock | Reduce copper(II) and can generate reactive oxygen species |
| Strongly acidic diluents | Avoid | Low pH releases copper from the complex |
Equipment and preparation
- KLOW vial and its lot-matched COA (see the COA library and KLOW stack purity testing)
- Bacteriostatic water, in date and within 28 days of first puncture
- Sterile 3 mL or 5 mL syringe for diluent transfer; U-100 syringes or calibrated pipettes for aliquots
- 70% isopropyl alcohol swabs, gloves, and a clean work surface or biosafety cabinet
- Labels, and low-protein-binding tubes if you plan to aliquot
Check the physical capacity of the vial before choosing a volume. A 3 mL vial cannot accept a 4 mL fill; in that case use 2–3 mL and adjust the math, or reconstitute and transfer to a larger sterile container.
Volume selection: calculating the working concentration
For every component, concentration (mg/mL) = component mass (mg) ÷ diluent volume (mL). The table applies that to the standard 50 / 10 / 10 / 10 mg composition. The right-hand columns show the mass of each component in 10 units (0.1 mL) on a U-100 laboratory syringe scale, where 100 units = 1 mL.
| Diluent added | GHK-Cu (mg/mL) | KPV (mg/mL) | BPC-157 (mg/mL) | TB-500 (mg/mL) | Total peptide (mg/mL) | GHK-Cu per 10 units | KPV per 10 units | BPC-157 per 10 units | TB-500 per 10 units |
|---|---|---|---|---|---|---|---|---|---|
| 2 mL | 25 | 5 | 5 | 5 | 40 | 2,500 mcg | 500 mcg | 500 mcg | 500 mcg |
| 3 mL | 16.67 | 3.33 | 3.33 | 3.33 | 26.67 | 1,667 mcg | 333 mcg | 333 mcg | 333 mcg |
| 4 mL | 12.5 | 2.5 | 2.5 | 2.5 | 20 | 1,250 mcg | 250 mcg | 250 mcg | 250 mcg |
| 5 mL | 10 | 2 | 2 | 2 | 16 | 1,000 mcg | 200 mcg | 200 mcg | 200 mcg |
| 8 mL | 6.25 | 1.25 | 1.25 | 1.25 | 10 | 625 mcg | 125 mcg | 125 mcg | 125 mcg |
How to choose:
- 4 mL is the most convenient general-purpose volume: 20 mg/mL total, with 2.5 mg/mL of each minor component (25 mcg per unit mark) and 12.5 mg/mL GHK-Cu (125 mcg per unit mark).
- 2–3 mL suits smaller vials or work that needs a concentrated stock. At 2 mL the solution is 40 mg/mL total; it will be a deeper blue and may take longer to clear.
- 5–8 mL gives finer volumetric resolution when small masses are measured, at the cost of more volume per aliquot. Eight milliliters yields a tidy 10 mg/mL total.
The reconstitution calculator will compute any other combination; enter the mass of the component you are tracking, not the 80 mg total, if you want a per-component figure.
Molar equivalents
| Component | Molar concentration per 1 mg/mL | At 4 mL fill |
|---|---|---|
| GHK-Cu (≈ 404 g/mol) | ≈ 2.48 mM | 12.5 mg/mL ≈ 31 mM |
| KPV (≈ 342 g/mol) | ≈ 2.92 mM | 2.5 mg/mL ≈ 7.3 mM |
| BPC-157 (≈ 1,419.5 g/mol) | ≈ 704 µM | 2.5 mg/mL ≈ 1.76 mM |
| TB-500 (≈ 4,963 g/mol) | ≈ 201 µM | 2.5 mg/mL ≈ 504 µM |
Equal mass is far from equal moles in this blend: at the same mg/mL, KPV is present at roughly fourteen times the molar concentration of TB-500. Values assume 100% peptide content; correct with COA data for exact work.
Step-by-step reconstitution protocol
- Equilibrate. Leave the sealed vial at room temperature for 15–30 minutes so condensation does not form inside when it is pierced.
- Inspect. The cake is normally pale blue to blue because of the copper complex. Confirm the seal is intact and the lot number matches the COA.
- Disinfect. Swab both stoppers with 70% isopropyl alcohol and allow them to dry.
- Draw the diluent. Pull the planned volume of bacteriostatic water into a sterile syringe.
- Add slowly. Insert the needle at an angle and let the water run down the inside wall. If the vial is under vacuum, control the plunger so the water does not jet onto the cake.
- Swirl gently. Roll the vial for 30–60 seconds. Do not shake or vortex; TB-500 in particular is a longer peptide that is better protected from foaming.
- Rest and inspect. Allow a few minutes for complete dissolution. The solution should be a clear, uniform blue with no particles.
- Label and refrigerate. Record date, diluent, volume and per-component concentrations, then store at 2–8 °C.
Reading the color
The blue color is expected and is a useful built-in indicator. A clear blue solution is normal. Turbidity, a precipitate, or a shift toward green, brown or colorless suggests copper has been displaced, reduced or precipitated, or that the solution is contaminated, and the vial should be set aside.
Storage of lyophilized and reconstituted KLOW
| State | Temperature | Working window | Notes |
|---|---|---|---|
| Lyophilized, long-term | −20 °C | 12–24+ months | Sealed, boxed, dry |
| Lyophilized, short-term | 2–8 °C | Up to a few months | Away from the refrigerator door |
| Lyophilized, shipping | Ambient | Days | Refrigerate or freeze on arrival |
| Reconstituted, bacteriostatic water | 2–8 °C | Up to about 28 days | Upright, dark; minimize time at room temperature |
| Reconstituted, sterile water | 2–8 °C | Same day | Discard remainder or aliquot and freeze immediately |
| Reconstituted aliquots | −20 °C or colder | 1–3 months | Low-bind tubes; thaw once |
In a blend, the least stable component and the microbiological limit jointly define the window. GHK-Cu and KPV are small, robust tripeptides. BPC-157 is known for good solution stability. TB-500, at 43 residues with a methionine, is the component most exposed to oxidation and aggregation, so the handling rules that protect it (cold, dark, no shaking, no repeated freeze–thaw) govern the whole vial. The 28-day figure is the preserved-container convention, not a measured shelf life for this combination; for sensitive assays, use fresher solution. Component-specific notes are in BPC-157 reconstitution and storage and TB-500 reconstitution and storage.
Dilution for cell culture and in vitro assays
Stock concentrations are far above typical in vitro working ranges, so serial dilution is required. Dilute from the water stock into assay buffer or medium immediately before use, and do not store diluted working solutions.
| Dilution from a 4 mL stock | GHK-Cu | KPV, BPC-157, TB-500 (each) | Benzyl alcohol carried over |
|---|---|---|---|
| Stock (undiluted) | 12.5 mg/mL | 2.5 mg/mL | 0.9% |
| 1:10 | 1.25 mg/mL | 250 µg/mL | 0.09% |
| 1:100 | 125 µg/mL | 25 µg/mL | 0.009% |
| 1:1,000 | 12.5 µg/mL | 2.5 µg/mL | 0.0009% |
| 1:10,000 | 1.25 µg/mL | 250 ng/mL | 0.00009% |
- Include a vehicle control. Benzyl alcohol can affect some cell types at higher concentrations, so match the diluent in control wells.
- Filter if sterility is critical. A 0.22 µm low-protein-binding (PES or PVDF) syringe filter can be used; expect some loss to the membrane and pre-wet the filter where possible.
- Remember the fixed ratio. Diluting the blend lowers all four concentrations together. Titrating one component independently requires separate vials, such as GHK-Cu, KPV, BPC-157 and TB-500.
- Mind copper in the medium. Media containing chelators or high levels of free histidine or albumin can redistribute copper away from GHK; note medium composition in the methods.
Common mistakes
- Recording 20 mg/mL as though it were the concentration of each peptide, when it is the total.
- Using a phosphate buffer or a chelator-containing solution as the stock diluent.
- Assuming the blue color means something is wrong, or ignoring a color change that does.
- Shaking the vial to hurry dissolution.
- Leaving the reconstituted vial on the bench between uses.
- Refreezing the multi-use vial instead of preparing single-use aliquots.
Summary
The KLOW stack is an 80 mg, four-peptide lyophilized blend dominated by GHK-Cu. Reconstitute it gently with bacteriostatic water, calculate each component separately, expect a clear blue solution, keep it at 2–8 °C in the dark, and work within about four weeks. For related reading, see the KLOW stack research FAQ, mechanism of action, and peptide blends and research stacks.
References
- United States Pharmacopeia. General Chapter <797> Pharmaceutical Compounding—Sterile Preparations. USP–NF.
- United States Pharmacopeia. General Chapter <51> Antimicrobial Effectiveness Testing. USP–NF.
- Pickart L, Margolina A. Regenerative and protective actions of the GHK-Cu peptide in the light of the new gene data. Int J Mol Sci. 2018;19(7):1987.
- Dalmasso G, Charrier-Hisamuddin L, Nguyen HTT, Yan Y, Sitaraman S, Merlin D. PepT1-mediated tripeptide KPV uptake reduces intestinal inflammation. Gastroenterology. 2008;134(1):166-178.
- Sikiric P, Seiwerth S, Rucman R, et al. Brain-gut axis and pentadecapeptide BPC 157: theoretical and practical implications. Curr Neuropharmacol. 2016;14(8):857-865.
- Goldstein AL, Hannappel E, Sosne G, Kleinman HK. Thymosin β4: a multi-functional regenerative peptide. Basic properties and clinical applications. Expert Opin Biol Ther. 2012;12(1):37-51.
- Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. Stability of protein pharmaceuticals: an update. Pharm Res. 2010;27(4):544-575.
- Meyer BK, Ni A, Hu B, Shi L. Antimicrobial preservative use in parenteral products: past and present. J Pharm Sci. 2007;96(12):3155-3167.
Frequently Asked Questions
What is the KLOW stack?
KLOW is a co-lyophilized laboratory blend of four synthetic peptides in one vial: GHK-Cu, KPV, BPC-157 and TB-500. The standard 80 mg vial contains 50 mg GHK-Cu and 10 mg of each of the other three. It is supplied for laboratory research only.
How much bacteriostatic water should I add to an 80 mg KLOW vial?
Four milliliters is the most common choice, giving 20 mg/mL total peptide, 12.5 mg/mL GHK-Cu and 2.5 mg/mL each of KPV, BPC-157 and TB-500. Other volumes work equally well as long as the vial can hold them and the concentration is recalculated.
Why is the reconstituted KLOW solution blue?
The color comes from copper(II) bound in the GHK-Cu complex. A clear blue solution is normal. Cloudiness, precipitate or a change toward green, brown or colorless indicates a problem.
How long does reconstituted KLOW last in the refrigerator?
In bacteriostatic water at 2–8 °C and protected from light, the conventional working window is up to about 28 days. This follows the standard for preserved multi-use containers rather than a stability study of the blend itself.
Can the ratio of peptides in the blend be changed?
No. The ratio is fixed when the vial is filled. Adding more or less water changes all four concentrations proportionally. Studies that need a different ratio should use individual vials of each peptide.
Can I reconstitute KLOW in saline or PBS?
It is better to make the stock in bacteriostatic or sterile water. Concentrated copper can precipitate with phosphate, and buffers offer no preservative protection. Use PBS or medium only for the final working dilution, prepared immediately before use.
What is the difference between KLOW and GLOW?
GLOW is the three-peptide blend of GHK-Cu, BPC-157 and TB-500, typically 70 mg per vial. KLOW adds 10 mg of KPV to make a four-peptide, 80 mg blend.
Can reconstituted KLOW be frozen?
Yes, as single-use aliquots in low-protein-binding tubes at −20 °C or colder, thawed once. Avoid repeatedly freezing and thawing the working vial.
How many units on a U-100 syringe scale equal 1 mg of total KLOW peptide at 20 mg/mL?
At 20 mg/mL, 1 mg of total peptide occupies 0.05 mL, which is 5 units on a U-100 scale. Those 5 units contain 625 mcg GHK-Cu and 125 mcg each of KPV, BPC-157 and TB-500.
Do I need to filter the solution?
Not for routine handling when sterile technique and bacteriostatic water are used. For cell-culture work where sterility is critical, a 0.22 µm low-protein-binding filter can be used, accepting a small loss of peptide to the membrane.