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PCK2 / Phosphoenolpyruvate carboxykinase [GTP], mitochondrial · Western blot design guide
Design a Western Blot for PCK2
Real validated PCK2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-PCK2 WB antibodies. Everything you need to plan the experiment before you commit precious samples.
Evidence assembled September 2026 · For research use; verify linked source records and product datasheet before use
PCK2 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 | ~70.7 kDa | |
| Observed band | ~71 kDa | |
| Gel | 5–20% (catalog M04772-2) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Adipose tissue (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | Modification-state controls | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 3 isoform(s) |
Section 1
Real Curated PCK2 Western Blot Protocols
The M04772-2 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human HEPG2, human Hela, human HEK293 (catalog M04772-2) |
| Gel % | 5–20% (catalog M04772-2) |
| Load | 50ug; reducing conditions (catalog M04772-2) |
| Transfer | a Nitrocellulose membrane at 150mA for 50-90 minutes (catalog M04772-2) |
| Membrane | Nitrocellulose membrane (catalog M04772-2) |
| Blocking | 5% Non-fat Milk/ TBS for 1.5 hour at RT (catalog M04772-2) |
| Primary antibody | M04772-2 · 0.25 μg/mL (catalog M04772-2) |
| Primary incubation | overnight at 4°C (catalog M04772-2) |
| Secondary antibody | goat anti-mouse IgG-HRP, 1:10000 (catalog M04772-2) |
| Secondary incubation | 1.5 hour at RT (catalog M04772-2) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M04772-2) |
| Detection | ECL (catalog M04772-2) |
Section 2
What Is the Expected PCK2 Western Blot Band Size?
PCK2 is predicted at 70.7 kDa and observed at ~71 kDa; isoforms are annotated, but distinct migration is not demonstrated.
| Band at ~71 kDa | Matches the empirical PCK2 band seen in reducing whole-cell and tissue lysates |
| Single band near 70.7 kDa | Consistent with the predicted mass of monomeric PCK2 |
| Several bands at different positions | Could reflect isoforms 1, 2, and 3 if their migration differs; band identities need confirmation |
| Weak whole-cell band that strengthens after mitochondrial enrichment | Consistent with PCK2 localization in mitochondria |
💡Expected PCK2 appearancePCK2 has a predicted mass of 70.7 kDa and an empirical band at ~71 kDa under reducing conditions; confirm band identity with appropriate controls.
| UniProt predicted mass | Places the reference monomer near 70.7 kDa |
| Splice isoform 1 | May differ in size from other isoforms; its individual mass is not supplied |
| Splice isoform 2 | May differ in size from other isoforms; its individual mass is not supplied |
| Splice isoform 3 | May differ in size from other isoforms; its individual mass is not supplied |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Mitochondrial PCK2 may be poorly represented in the sampled lysate | Check mitochondrial recovery and run a PCK2-positive control |
| Band higher than expected | The band may be an unverified isoform or nonspecific signal | Compare with a positive control and validate band identity |
| Band lower than expected | An isoform or protein breakdown could account for the band | Check sample integrity and validate band identity |
| Multiple bands | Isoforms 1, 2, and 3 are annotated, but their migration is unknown | Compare bands with isoform-specific controls |
| Weak or no signal | Whole-cell sampling may yield little mitochondrial PCK2 | Check mitochondrial recovery and include a positive control |
| Fragments below expected size | Protein breakdown during sample handling is possible | Prepare fresh lysate with protease inhibitors and compare with a positive control |
Experiment evidence
Western blot images showing PCK2 band
M04772-2 · PCK2
A04772-1 · PCK2
M04772 · PCK2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for PCK2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for PCK2 in Western blot, you can use appendix tissue, which HPA scores High.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Adipose tissue (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain such as Ponceau alongside the samples.
⚠️Feasibility: Adipose tissue is a feasible negative candidate, though HPA non-detection does not guarantee an absent Western-blot signal.
HPA tissue expression evidence for PCK2
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.
Higher expression tissues · candidate positive controls from IHC
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | endocrine cells | High | Protein (IHC) | HPA → |
| Colon | endocrine cells | High | Protein (IHC) | HPA → |
| Duodenum | endocrine cells | High | Protein (IHC) | HPA → |
| Kidney | proximal tubules (cell body) | High | Protein (IHC) | HPA → |
| Lung | alveolar cells type II | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PCK2 Western Blot Tips
Deeper troubleshooting and optimisation questions for PCK2, answered from its protein features.
How should PCK2 band migration be interpreted?
Band shift · Use the separately labelled calculated mass and catalog-observed evidence above. A sequence annotation does not establish an observed migration shift. Verify target identity with orthogonal controls.
Could PCK2 isoforms produce different bands?
Isoforms · UniProt lists three isoforms. Isoform 3 lacks canonical residues 1–134, while isoform 2 replaces canonical residues 413–640 with a shorter sequence. Either sequence change could affect apparent size; the supplied features do not establish where those isoforms migrate on a blot.
Check the antibody’s binding region against the listed sequence changes. Canonical residues 1–134 are absent from isoform 3, and residues 413–640 are replaced in isoform 2. An antibody targeting either region may detect the isoforms differently.
Check the antibody’s binding region against the listed sequence changes. Canonical residues 1–134 are absent from isoform 3, and residues 413–640 are replaced in isoform 2. An antibody targeting either region may detect the isoforms differently.
Which PCK2 modifications matter when interpreting bands?
PTM · UniProt lists phosphothreonine at 42 and 196, phosphoserine at 115 and 304, N6-acetyllysine at 88, and N6-succinyllysine at 457. These are canonical UniProt coordinates; antibody or paper numbering may differ. Their presence alone does not establish a visible band shift.
Does this guide establish induction of PCK2?
Induction · No general induction response is established by this guide. A pathway or gene-set association is not evidence of induction in a particular specimen. Verify the relevant treatment and control in a target-specific experiment.
How should transfer be checked for PCK2?
Transfer · Standard workflow guidance: verify transfer efficiency for the intended target size before interpreting a weak signal. Use total-protein assessment and optimize transfer for the membrane, gel and apparatus; the labelled catalog values take precedence.
How should blocking be optimized?
Blocking · Standard workflow guidance: follow the M04772-2 datasheet where specified. Otherwise compare 5% milk or 5% BSA in TBST; for a phospho-specific assay start with BSA. Optimize background and specific signal with matched controls.
How should I quantify a PCK2 Western blot?
Quantitation · Quantify the same assigned PCK2 band across comparable samples, accounting for the three listed isoforms if the antibody detects more than one. Use consistent sample preparation because PCK2 is mitochondrial. A change in one band alone does not establish a change in total PCK2 across isoforms.
Why does PCK2 appear near 71 kDa?
Interpretation · The observed band near 71 kDa agrees with the 70.7 kDa predicted mass of canonical PCK2. The listed modifications and alternative sequences do not, by themselves, establish a visible shift or explain a mass difference.
PCK2 is annotated in mitochondria. Include a mitochondria-containing sample when assessing its band, and keep sample preparation consistent across lanes. The supplied features do not establish how much PCK2 any particular sample contains.
First compare them with the canonical band near 71 kDa and consider the documented isoform 3 deletion and isoform 2 replacement. Antibody binding regions can help assess which isoforms are detectable. The listed modifications do not alone identify an unexpected band or prove that it is PCK2.
PCK2 is annotated in mitochondria. Include a mitochondria-containing sample when assessing its band, and keep sample preparation consistent across lanes. The supplied features do not establish how much PCK2 any particular sample contains.
First compare them with the canonical band near 71 kDa and consider the documented isoform 3 deletion and isoform 2 replacement. Antibody binding regions can help assess which isoforms are detectable. The listed modifications do not alone identify an unexpected band or prove that it is PCK2.
Boster reagents
PCK2 Western Blot Antibodies
Catalog antibodies with Western blot application and product-specific WB images. Evaluate suitability with the reported sample, controls and experimental conditions.
Real WB data
Anti-PCK2 Antibody Picoband® (monoclonal, 3F7)
Cat # M04772-2
Real WB data
Anti-PCK2 Antibody Picoband®
Cat # A04772-1
Real WB data
Anti-PCK2 Monoclonal Antibody
Cat # M04772
Three the supplier anti-PCK2 antibodies have Western blot images showing a band near the expected 71 kDa in the listed lysates. These product images document tested samples and conditions; independent validation is not supplied.
Which to pick: For an image matching your species, choose M04772-2 for human, monkey, rat, or mouse samples; A04772-1 for human or monkey samples; or M04772 for human, rat, or mouse samples. Check the caption for the closest tissue or cell type.
Source: BosterBio PCK2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q16822.
- Human Protein Atlas. PCK2 tissue expression.
- PMC12948644 — target-verified WB comparison
- PMC11189538 — target-verified WB comparison
- PMC9379418 — target-verified WB comparison