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RACGAP1 / Rac GTPase-activating protein 1 · Western blot design guide
Design a Western Blot for RACGAP1
Source-linked RACGAP1 Western blot protocol options, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RACGAP1 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
RACGAP1 Western Blot Experimental Design Guide
Expected bands, source-linked protocol options, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before running
| Expected band | ~71 kDa | |
| Observed band | ~71 kDa | |
| Gel | 10% (catalog M03018-1) | |
| 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 | Phosphorylation-state controls | |
| Gene-set association | MSigDB Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Source-Linked RACGAP1 Western Blot Protocol Options
The M03018-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human MCF-7, human SIHA, human Hela, human A549, human Caco-2, human U20S, human PC-3 (catalog M03018-1) |
| Gel % | 10% (catalog M03018-1) |
| Load | 30 ug; reducing conditions (catalog M03018-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M03018-1) |
| Membrane | nitrocellulose membrane (catalog M03018-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M03018-1) |
| Primary antibody | M03018-1 · 1:500 (catalog M03018-1) |
| Primary incubation | overnight at 4°C (catalog M03018-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog M03018-1) |
| Secondary incubation | 1.5 hour at RT (catalog M03018-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M03018-1) |
| Detection | ECL (catalog M03018-1) |
Section 2
What Is the Expected RACGAP1 Western Blot Band Size?
RACGAP1 is predicted at 71 kDa and observed near 71 kDa; its annotated modifications have no demonstrated effect on migration here.
| Band near 71 kDa | Consistent with the predicted mass and the observed RACGAP1 band; confirm identity with controls |
| Weak band in soluble lysate | RACGAP1 may be retained in cytoskeletal or membrane-associated fractions |
| Close doublet near 71 kDa | Phosphorylation is possible, but distinct migration has not been demonstrated |
| Additional higher band | Its identity is uncertain; the annotated heterotetramer does not establish a higher SDS-PAGE band |
💡Expected RACGAP1 appearanceRACGAP1 has a predicted mass of 71 kDa and an empirical band at approximately 71 kDa in reducing whole-cell lysates; confirm band identity with ordinary antibody controls.
| Predicted RACGAP1 mass | Places the full-length protein near 71 kDa |
| N-acetylmethionine at residue 1 | Has no demonstrated effect on apparent band size |
| PLK1 phosphoserines at residues 149, 157, 164 and 170 | Could affect migration, but no shift is established |
| Phosphoserines at residues 154 and 203 and phosphothreonine at residue 161 | Could affect migration, but no shift is established |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | RACGAP1 may partition with cytoskeletal or membrane-associated material | Check the insoluble fraction and verify extraction |
| Band higher than expected | Identity or modification state is unconfirmed | Compare reducing samples and verify the band with an independent antibody or RACGAP1 depletion |
| Band lower than expected | A smaller band is not explained by an annotated signal peptide or propeptide | Check sample integrity and confirm identity with RACGAP1 depletion |
| Multiple bands | Phosphorylation is annotated, but separate bands are not established | Compare phosphatase-treated samples and verify each band independently |
| Weak or no signal | Recovery may vary with RACGAP1 localization to the cytoskeleton and cell structures | Check extraction, loading and antibody performance with a positive lysate |
Experiment evidence
Western blot images showing RACGAP1 band
M03018-1 · RACGAP1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RACGAP1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for RACGAP1 in Western blot, you can use appendix tissue, which HPA rates as highly expressed.
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 alongside the samples.
⚠️Feasibility: HPA supports appendix as a positive and adipose tissue as a negative, making tissue controls feasible.
HPA tissue expression evidence for RACGAP1
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 | germinal center cells | High | Protein (IHC) | HPA → |
| Duodenum | glands of Brunner | High | Protein (IHC) | HPA → |
| Testis | pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Bone marrow | hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Cervix | squamous epithelial cells | Medium | 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 → |
| Breast | adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | basal cells | Not detected | Protein (IHC) | HPA → |
| Caudate | glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RACGAP1 Western Blot Tips
Deeper troubleshooting and optimisation questions for RACGAP1, answered from its protein features.
How should RACGAP1 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 RACGAP1 isoforms explain multiple bands?
Isoforms · The supplied record lists one isoform and no alternative sequence. It does not support assigning additional bands to known isoforms.
Which PLK1 phosphorylation sites matter when comparing bands?
PTM · UniProt lists PLK1 phosphorylation at Ser149, Ser157, Ser164, and Ser170. These are UniProt coordinates; check the numbering used by any antibody or paper. Their presence does not establish a visible band shift.
Yes. UniProt lists AURKB phosphorylation at Ser387 and Ser410, using UniProt coordinates. These sites may guide phospho-specific antibody selection, but the feature list does not show how they affect migration.
Yes. UniProt lists AURKB phosphorylation at Ser387 and Ser410, using UniProt coordinates. These sites may guide phospho-specific antibody selection, but the feature list does not show how they affect migration.
Does this guide establish induction of RACGAP1?
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 RACGAP1?
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 M03018-1 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 RACGAP1 be quantified?
Quantitation · Standard workflow guidance: quantify only a validated target band within the linear exposure range. Use consistent sample preparation and loading, retain biological replicates, and avoid interpreting saturation or loading differences as regulation.
Should RACGAP1 run above its predicted mass?
Interpretation · Its predicted mass is 71 kDa, matching the reported band near 71 kDa. The listed modifications alone do not establish a visible shift or explain any mass difference.
RACGAP1 has cell-cycle and cell-division annotations and localizes to the spindle, cleavage furrow, and midbody. Record cell-cycle conditions when comparing samples, and use the same sample preparation and loading approach for quantitation. The supplied features do not establish induction of total RACGAP1.
RACGAP1 is listed in the nucleus, cytoplasm, cytoskeleton, and at the cytoplasmic side of the cell membrane. Compare like fractions when quantifying its band; differences between fractions need not reflect a change in total protein.
The record lists phosphorylation sites and N-acetylmethionine at position 1, but these features alone cannot identify an unexpected band. One isoform is listed, and no glycosylation sites are supplied. Compare the extra bands with the expected band near 71 kDa before assigning an identity.
RACGAP1 has cell-cycle and cell-division annotations and localizes to the spindle, cleavage furrow, and midbody. Record cell-cycle conditions when comparing samples, and use the same sample preparation and loading approach for quantitation. The supplied features do not establish induction of total RACGAP1.
RACGAP1 is listed in the nucleus, cytoplasm, cytoskeleton, and at the cytoplasmic side of the cell membrane. Compare like fractions when quantifying its band; differences between fractions need not reflect a change in total protein.
The record lists phosphorylation sites and N-acetylmethionine at position 1, but these features alone cannot identify an unexpected band. One isoform is listed, and no glycosylation sites are supplied. Compare the extra bands with the expected band near 71 kDa before assigning an identity.
Boster reagents
RACGAP1 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-RGAP1 Rabbit Monoclonal Antibody
Cat # M03018-1
The catalog reports one anti-RACGAP1 antibody, M03018-1, with a Western blot image showing an approximately 71 kDa band in seven human whole-cell lysates. No publication evidence or nonhuman WB data are supplied.
Which to pick: M03018-1 is the only listed option. Its reported human reactivity and WB image make it the documented choice for human samples; the caption reports a 1:500 primary dilution and 30 µg of lysate per lane.
Source: BosterBio RACGAP1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry Q9H0H5.
- Human Protein Atlas. RACGAP1 tissue expression.
- PMC11018837 — target-verified WB comparison
- PMC12729191 — target-verified WB comparison
- PMC10529746 — target-verified WB comparison