Western Blot — Serine Proteinases

Serine proteinases are the largest proteinase class in the human proteome — over 170 members — spanning secreted enzymes, membrane-anchored enzymes, and intracellular proteases. Major clinically-relevant subfamilies include: granzymes (immune cell cytotoxicity), kallikreins (KLK1-15, tissue kallikreins including PSA), TMPRSS family (TMPRSS2, TMPRSS4, and related type II membrane serine proteases), coagulation cascade factors (factor VII, IX, X, XI, XII), complement proteases (C1r, C1s, MASP1, MASP2, factor B, factor D), the digestive enzymes (trypsin, chymotrypsin, elastase), and neutrophil-derived enzymes (neutrophil elastase, cathepsin G, proteinase 3).

This protocol covers Western blot detection of serine proteinases with Triple Point rabbit polyclonal antibodies. Standard mechanics are covered in other protocols; this document focuses on the serine-proteinase-specific considerations.

Sample preparation — the class-mate cross-cleavage problem

Serine proteinases in samples are often accompanied by other serine proteinases. Neutrophil-derived samples contain neutrophil elastase, cathepsin G, and proteinase 3 in the same granule; pancreatic samples contain trypsin, chymotrypsin, and elastase in the same secretion. During sample preparation, one serine proteinase can cleave another — and the antibody target's apparent MW will be reduced by this cross-cleavage.

The primary defence is to include class-specific serine proteinase inhibitors in the lysis buffer:

A defensive cocktail: AEBSF (100 μM) + aprotinin (2 μg/mL) + leupeptin (10 μM) + 1× complete protease inhibitor cocktail. This blocks nearly all classes of serine proteinase without inhibiting the antibody's ability to bind target protein — you can add class-appropriate inhibitors for other classes (E-64 for cysteine, pepstatin for aspartic, 1,10-phenanthroline for metallo) alongside without conflict.

Expected molecular weights — zymogen vs mature, glycosylation shifts

Serine proteinases are almost universally synthesised as zymogens with an N-terminal propeptide. Activation involves cleavage of a specific bond that generates a new N-terminus (often starting with Ile16 in the chymotrypsin numbering scheme), and this new N-terminus forms a salt bridge with Asp194 (chymotrypsin numbering) that stabilises the catalytic conformation. The MW difference between zymogen and mature is small (typically 1-3 kDa — just the propeptide) and may or may not be resolvable on a standard gel.

Glycosylation shifts, however, are large. Many serine proteinases have multiple N-glycosylation sites, and mature glycosylated forms can run 5-20 kDa above the calculated protein MW.

Serpin complexes — the SDS-stable complex problem

Serpins (serine proteinase inhibitors) form covalent SDS-stable acyl-enzyme complexes with their target serine proteinases. Unlike TIMP-MMP complexes (which are non-covalent and dissociate under some conditions), serpin-serine proteinase complexes survive SDS denaturation and reduction. They appear on Western blots as high-MW bands equal to the sum of the serpin MW plus the proteinase MW.

For example:

• Thrombin-antithrombin complex (TAT): ~93 kDa (thrombin 37 kDa + antithrombin 58 kDa, minus a leaving peptide)
• Neutrophil elastase-alpha-1-antitrypsin complex: ~80 kDa (elastase 29 kDa + AAT 52 kDa)
• Plasmin-alpha-2-antiplasmin complex: ~150 kDa

If your Western blot shows a high-MW band at the sum of the target MW plus a known cognate serpin MW, it may be a serpin complex — not degradation product, not aggregation, but legitimate biology. Confirm by co-probing for the cognate serpin on the same blot.

Gel and transfer conditions

Reducing SDS-PAGE with sample boiling at 95°C for 10 minutes in Laemmli buffer with 5% β-mercaptoethanol or 100 mM DTT is standard for all serine proteinase Western blots. For granzyme A specifically, ensure full reduction — the disulfide-linked homodimer resists partial reduction.

Gel percentage: 10% for most serine proteinases (25-55 kDa range). 8% for high-MW zymogens like prothrombin. 12% for small mature enzymes like the granzymes. Gradient gels (4-20%) accommodate the full range if you are probing multiple targets.

Transfer: standard wet transfer 100V for 60 minutes covers the standard range. Membrane choice: PVDF for hydrophobic targets like TMPRSS-family membrane proteins; nitrocellulose acceptable for secreted enzymes.

Blocking and antibody incubation

5% milk in TBS-T (0.1% Tween-20). Triple Point serine proteinase antibodies at 1:1,000 to 1:5,000 per CoA, overnight at 4°C. Anti-rabbit HRP at 1:10,000, 1 hour room temperature. Standard ECL detection is usually sufficient for granzymes (highly expressed in cytotoxic lymphocytes), kallikreins (moderate expression in specific tissues), and TMPRSS family (moderate to low expression). Enhanced ECL for low-abundance targets.

Detection strategies for specific questions

"Is this serine proteinase activated?"

Two approaches:

• Propeptide + catalytic domain antibody pair. Propeptide antibody detects only zymogen. Catalytic-domain antibody detects both. Loss of propeptide signal + retention of a slightly-lower-MW catalytic-domain band = activation. The MW shift is small (1-3 kDa) so use a well-resolved gel.
• Activation-site-specific antibody. Some vendors sell antibodies that only detect the mature enzyme after propeptide cleavage — the epitope includes the newly-generated N-terminus (Ile16 or equivalent). If available, these are unambiguous readouts of activation state.

"Is this serine proteinase in a serpin complex?"

Probe cognate serpin and target proteinase on the same blot. Complex MW = target MW + serpin MW (approximately). A band at that predicted MW that is detected by both antibodies is the complex. This is a definitive assignment — two independent antibodies detecting the same band is stronger evidence than either alone.

"Is this membrane-anchored serine proteinase shed?"

For TMPRSS family and other type II membrane serine proteases, probe cell lysate and conditioned media. TMPRSS2, for example, autoactivates by cleavage between the transmembrane region and catalytic domain, releasing a soluble catalytic fragment into the media. Cell lysate shows both full-length pro-form and the small membrane-retained N-terminal fragment; media shows only the shed catalytic fragment. Different antibodies are needed for the cytoplasmic vs catalytic domains — check the CoA for which region each product targets.

Superpooled kits for serine proteinases

Triple Point Superpooled kits are available for granzymes A, B, and K, kallikreins 1-15 (individually), TMPRSS2, TMPRSS4, TMPRSS6, and selected coagulation factors. Each kit contains propeptide, catalytic-domain, and (for membrane-anchored targets) cytoplasmic-tail-directed polyclonals. See the SPA method overview and usage protocol.

Common failure modes specific to serine proteinases

Degradation ladder from neutrophil samples

Neutrophil elastase, cathepsin G, and proteinase 3 in the same lysate cross-cleave each other. Use the full defensive inhibitor cocktail (AEBSF + aprotinin + leupeptin), keep cold, boil in sample buffer immediately.

Serpin complex misidentified as aggregation

High-MW bands above expected zymogen MW may be serpin complexes, not aggregates. Check the CoA for known cognate serpins and probe both on the same blot to confirm.

Granzyme A dimer confusion under non-reducing conditions

Granzyme A is a disulfide-linked homodimer. Under non-reducing gels it runs at ~55 kDa (not 27 kDa monomer). Always use fully-reducing sample buffer.

Kallikrein cross-reactivity

Kallikreins 1-15 share substantial sequence identity within the family. Triple Point's individual kallikrein antibodies are raised against divergent peptide immunogens; cross-reactivity is measured and reported on the CoA. If cross-reactivity is a concern, run adjacent lanes with paralogue-specific detection.

TMPRSS2 detection failing in COVID research context

TMPRSS2 is a proteinase of interest in SARS-CoV-2 research. It is highly N-glycosylated and its apparent MW varies with cell type. Run PNGase F control to confirm identity by MW collapse. See individual product datasheet for the specific expected MW in your cell line.

PSA detection in serum vs prostate tissue

Serum PSA is predominantly the PSA-ACT (alpha-1-antichymotrypsin) complex at ~90 kDa. Prostate tissue PSA is predominantly free at ~33 kDa. If you are running serum-derived samples, do not look for signal at 33 kDa — the free form is a minor fraction.

Positive control samples for serine proteinases

Related

• Serine proteinase antibody catalog
• Granzyme A vs granzyme B selection
• Granzyme B vs granzyme K selection
• MASP1 vs MASP2 selection
• The Superpooled (SPA) method
• Dilution optimization
• Controls and validation
• IP protocol for proteinase enrichment
• Zymogens and proenzymes overview