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GABARAP · Western blot design guide
Design a Western Blot for GABARAP
Real validated GABARAP Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-GABARAP WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Last reviewed: May 2026 · Scientific review: Boster Bio technical team
GABARAP Western Blot Experimental Design Guide
Expected bands, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~13.9 kDa | |
| Gel | 12–15% | |
| Negative control | siRNA / KO lysate |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Cleaved | |
| Caveat | Lipidation alters mobility | |
| Regulation | IFN-γ-induced | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated GABARAP Western Blot Protocols
Literature-validated Western blot parameters for GABARAP — gel percentage, transfer, blocking, antibody incubation and detection, extracted from published methods.
| Sample / lysate | human HEK293 , Lane 2: human Jurkat , Lane 3: human A549 , Lane 4: human HELA , Lane 5: human U87 , Lane 6: human HEPG2 , Lane 7: human U937 , Lane 8: rat liver , Lane 9: rat pancreas , Lane 10: mouse liver , Lane 11: mouse pancreas . After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-GABARAP antigen affinity purified polyclonal antibody (Catalog # A01907-2) at 0.25 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for GABARAP at approximately 18KD. The expected band size for GABARAP is at 18KD |
| Gel % | 12–15% |
| Load | 50ug |
| Transfer | nitrocellulose membrane, 150 mA, 50–90 min |
| Membrane | nitrocellulose |
| Blocking | 5% non-fat milk / TBS, 1.5 h RT |
| Primary antibody | 0.25 µg/mL |
| Primary incubation | overnight at 4 °C |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 |
| Wash | TBS-0.1% Tween, 3 × 5 min |
| Detection | ECL |
| Exposure / imaging | Tanon 5200 |
| Observed band | 18 kDa |
Section 2
What Is the Expected GABARAP Western Blot Band Size?
GABARAP's 13.9 kDa predicted mass should appear as a near-identical ~14 kDa band, with only a minor shift expected from C-terminal propeptide processing to the mature form.
| single band near 13-14 kDa | matches the predicted mass of unmodified, non-glycosylated 117-aa GABARAP with no disulfide-linked modification |
| band running just below the mass of the 117-aa full-length sequence | C-terminal propeptide cleavage at residue 117 generates the mature, processed form of GABARAP |
| closely spaced doublet near 14 kDa | co-occurrence of the uncleaved precursor and the propeptide-processed mature GABARAP species |
| band present across most whole-cell lysate lanes tested | cytoplasmic and membrane/vesicle-associated localization (autophagosome, Golgi, endomembrane) means GABARAP is retained intracellularly rather than secreted, so it is detectable directly in lysate |
| higher-molecular-weight band or smear above the monomer under mild denaturation | incomplete dissociation of GABARAP from interaction partners such as ATG7, ATG13, ATG3, GPHN, or NSF |
| no extra bands beyond the single species or doublet | only one isoform is annotated for GABARAP, so additional bands are not expected from alternative splicing |
💡Expected GABARAP appearanceExpect a single, sharp band around 13-14 kDa, matching the unmodified, non-glycosylated 117-residue GABARAP, with only a marginal downward shift from C-terminal propeptide cleavage to the mature form.
| predicted mass (13.9 kDa, 117 aa) | sets the baseline expected migration position near 13-14 kDa for the unmodified protein |
| C-terminal propeptide cleavage (residue 117) | removes the terminal propeptide to yield the mature form, giving a band marginally smaller than the full 117-aa precursor |
| single annotated isoform (no alternative splicing) | no isoform-derived bands are expected; any extra bands point to processing state or degradation rather than splice variants |
| membrane/vesicle-associated subcellular pool (autophagosome, Golgi, endomembrane, cytoskeleton) | partitioning into membrane and cytoskeletal fractions means incomplete lysis or solubilization can weaken apparent band intensity without changing its size |
| interactions with ATG7, ATG13, ATG3, GPHN, and NSF | under incomplete denaturation these complexes can co-migrate, producing higher-molecular-weight bands above the monomer |
| Situation | Likely cause | Next action |
|---|---|---|
| Band lower than expected | C-terminal propeptide cleavage removes the terminal residue to generate the mature, slightly smaller GABARAP species | confirm the antibody epitope location relative to the processed C-terminus and compare migration against a full-length recombinant standard |
| Band higher than expected | incomplete denaturation leaves GABARAP complexed with interacting partners such as ATG7, ATG13, ATG3, GPHN, or NSF | boil samples longer in fresh reducing SDS sample buffer to fully dissociate protein complexes before loading |
| Broad smear instead of sharp band | the small ~14 kDa size of GABARAP is poorly resolved on standard low-percentage gels | run a higher-percentage or gradient gel (e.g. 12-15% or 5-20%) to sharpen resolution of this low-molecular-weight protein |
| Multiple bands | co-existence of the uncleaved 117-aa precursor with the propeptide-processed mature form produces a closely spaced doublet | extend run time or use a higher-resolution low-MW gel system to separate the doublet and identify which species the antibody detects |
| Weak or no signal | small proteins like GABARAP can transfer through the membrane during blotting, or membrane/cytoskeleton-associated pools can be under-extracted | shorten transfer time or reduce voltage, use a 0.2 micron membrane, and use a lysis buffer that solubilizes membrane and cytoskeletal fractions |
Experiment evidence
Western blot images showing GABARAP band
A01907-2 · GABARAP
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for GABARAP Western blot
🧪For positive controls for GABARAP in Western blot, you can use whole-cell lysate from a common human cell line such as HeLa or HEK293, since GABARAP is a ubiquitously expressed autophagosome membrane protein and HPA does not provide tissue-specific expression data for this gene.
Positive control: HeLa or HEK293 cell lysate
Negative control: ubiquitously expressed; use siRNA knockdown or KO line
Loading controls: Run GAPDH and β-actin alongside a total-protein stain (e.g., stain-free gel, Ponceau S, or REVERT) as loading controls.
⚠️Feasibility: GABARAP localizes broadly to autophagosome and endomembrane compartments in most cell types, and no HPA-confirmed negative tissue is available, so a siRNA knockdown or CRISPR KO line is needed to establish a true negative control.
Comprehensive Human Protein Atlas IHC scoring per tissue. Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|
Section 3
Advanced GABARAP Western Blot Tips
Deeper troubleshooting and optimisation questions for GABARAP, answered from its protein features.
Why does GABARAP run as a doublet near its predicted mass?
GABARAP is proteolytically processed at Gly116 and lipidated with phosphatidylethanolamine, producing a faster-migrating lipidated form (GABARAP-II) alongside the unlipidated cytosolic form (GABARAP-I). Both run near the 13.9 kDa predicted mass, but GABARAP-II typically migrates slightly faster on standard SDS-PAGE, so expect two closely spaced bands rather than a single sharp band.
Does GABARAP have multiple isoforms explaining extra bands?
No; UniProt lists only a single GABARAP isoform, so additional bands are not due to alternative splicing. Extra bands more likely reflect the lipidation-based GABARAP-I/II doublet, degradation products, or cross-reactivity with the closely related GABARAPL1, GABARAPL2, or LC3 paralogs rather than distinct isoforms of GABARAP itself.
Does autophagy induction change the GABARAP band pattern?
Yes; GABARAP lacks annotated modified residues but undergoes lipidation upon autophagy induction (e.g., starvation), increasing the proportion of the faster-migrating lipidated GABARAP-II form relative to GABARAP-I. Comparing band intensity ratios before and after induction is a common way to monitor autophagic activity in these blots.
What blocking approach works best for this small protein?
Because GABARAP has no glycosylation and no disulfide bonds, standard 5% non-fat milk or BSA in TBST is sufficient; avoid harsh blocking that could obscure a small ~14 kDa signal. Since GABARAP is cytoplasmic and membrane-associated, ensure the antibody can access lipidated forms retained near the dye front.
What transfer method to use for GABARAP Western blot?
Use a low-percentage retention strategy: run a 12-15% or gradient gel and transfer to PVDF (better small-protein retention than nitrocellulose) at low voltage for a short time to prevent GABARAP, at ~13.9 kDa, from blotting through the membrane.
How should GABARAP-II be quantified relative to GABARAP-I?
Because GABARAP-II arises from lipidation of GABARAP-I, densitometric ratio of GABARAP-II to GABARAP-I (or to a loading control) is the standard readout for autophagic flux; pairing with lysosomal inhibitor treatment distinguishes increased flux from decreased degradation.
Could bands above 14 kDa indicate cross-reactivity?
GABARAP is highly homologous to GABARAPL1, GABARAPL2, and the LC3 family, so antibodies may cross-react, producing extra bands near the expected mass; bands substantially larger likely reflect non-specific binding or incomplete denaturation rather than GABARAP itself, given no disulfide bonds or glycosylation are annotated.
Boster reagents
Best GABARAP Western Blot Antibodies
BosterBio's GABARAP antibodies are among the best-performing WB antibodies on the market — well cited, thoroughly validated, and orthogonally cross-validated against negative tissues and complementary methods.
Real WB data
Anti-GABARAP Antibody Picoband®
Cat # A01907-2
Our recommended anti-GABARAP antibody is a best-performing, extensively cited reagent, rigorously validated by orthogonal methods including negative-tissue controls and complementary techniques, ensuring specific, reproducible detection in Western blot applications.
Which to pick: Only one Boster GABARAP antibody is catalogued, A01907-2; it includes an actual Western blot validation image, so it's the clear choice for GABARAP WB experiments.
Source: BosterBio GABARAP gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry O95166.
- Human Protein Atlas. GABARAP tissue expression.