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- Table of Contents
RACK1 / Small ribosomal subunit protein RACK1 · Western blot design guide
Design a Western Blot for RACK1
Real validated RACK1 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-RACK1 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
RACK1 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 | ~35.1 kDa | |
| Observed band | ~35 kDa | |
| Gel | 5–20% (catalog M02145-1) | |
| Positive control | Cerebellum (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 Hallmark membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated RACK1 Western Blot Protocols
The M02145-1 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 Jurkat, human HepG2 (catalog M02145-1) |
| Gel % | 5–20% (catalog M02145-1) |
| Load | 30 ug; reducing conditions (catalog M02145-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog M02145-1) |
| Membrane | nitrocellulose membrane (catalog M02145-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog M02145-1) |
| Primary antibody | M02145-1 · 1:500 (catalog M02145-1) |
| Primary incubation | overnight at 4°C (catalog M02145-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:1000 (catalog M02145-1) |
| Secondary incubation | 1.5 hour at RT (catalog M02145-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog M02145-1) |
| Detection | ECL (catalog M02145-1) |
Section 2
What Is the Expected RACK1 Western Blot Band Size?
RACK1 is predicted at 35.1 kDa and observed near 35 kDa; its annotated modifications and oligomerization do not establish additional migrating bands.
| Band near 35 kDa | Matches the observed RACK1 band and its 35.1 kDa predicted mass. |
| Band near 70 kDa | Could reflect a RACK1 homodimer if the complex survives sample preparation; verify its identity. |
| Bands above 35 kDa | Could reflect persistent RACK1 homooligomers, but their migration is not established. |
| Little or no 35 kDa band in lysate | Unexpected for cytoplasmic RACK1; check detection and sample quality. |
💡Expected RACK1 appearanceRACK1 is predicted at 35.1 kDa and observed near 35 kDa in reducing whole-cell lysates; confirm band identity with an appropriate antibody specificity control.
| 35.1 kDa predicted mass | Sets the approximate monomer band position, consistent with the observed 35 kDa band. |
| Homodimer and homooligomer formation | Could yield higher bands if complexes persist during electrophoresis; this is not demonstrated by the supplied blot. |
| N-terminal processing and acetylation | Are documented modifications, but a resolvable size change is not established. |
| Threonine and tyrosine phosphorylation | Is documented at specific residues, but a distinct migrating band is not established. |
| Lysine acetylation | Is documented at residues 130 and 183, but a distinct migrating band is not established. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Unexpected for cytoplasmic RACK1; sample or detection may have failed. | Check lysate integrity, loading, transfer, and a positive control. |
| Band higher than expected | A RACK1 complex might persist, although its gel migration is unproven. | Compare denaturing conditions and confirm band identity with an independent antibody or RACK1 depletion. |
| Band lower than expected | No signal peptide or propeptide cleavage explains a substantially smaller band. | Check sample integrity and confirm identity with an independent antibody or RACK1 depletion. |
| Multiple bands | Additional bands are not explained by distinct annotated isoforms; persistent complexes or unrelated antibody binding are possible. | Compare denaturing conditions and use RACK1 depletion to identify specific bands. |
| Weak or no signal | Insufficient RACK1 signal or inefficient detection may obscure the expected 35 kDa band. | Check loading, transfer, antibody performance, and a positive control. |
Experiment evidence
Western blot images showing RACK1 band
M02145-1 · RACK1
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for RACK1 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for RACK1 in Western blot, you can use cerebellum lysate, which HPA rates High.
Positive control: Cerebellum (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 lists adipose tissue as not detected, but verify the negative result in your lysate.
HPA tissue expression evidence for RACK1
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 |
|---|---|---|---|---|
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cervix | squamous epithelial cells | High | Protein (IHC) | HPA → |
| Colon | glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | glandular cells | High | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | 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 → |
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Liver | cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | myocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RACK1 Western Blot Tips
Deeper troubleshooting and optimisation questions for RACK1, answered from its protein features.
How should RACK1 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 RACK1 isoforms explain multiple bands?
Isoforms · The supplied features list one isoform and no alternative sequence. They do list N-terminally processed RACK1 with acetylthreonine at UniProt position 2, as well as acetylmethionine at position 1. Do not assign additional bands to isoforms from these features alone.
Which RACK1 modifications matter when interpreting band changes?
PTM · Listed sites include phosphothreonine at UniProt positions 6, 10, 96 and 316; phosphotyrosine at 52 and 228; N6-acetyllysine at 130 and 183; and phosphorylation at 276–279. These features identify possible molecular differences, but do not establish that any produces a visible band shift. Keep UniProt numbering explicit when comparing antibody or paper annotations.
The features assign phosphoserine 276, phosphothreonine 277, phosphoserine 278 and phosphoserine 279 to viral VacV B1 kinase. If comparing VacV-exposed samples, consider these sites when interpreting a phosphorylation-sensitive signal. Their listing does not establish a detectable shift on a standard blot.
The features assign phosphoserine 276, phosphothreonine 277, phosphoserine 278 and phosphoserine 279 to viral VacV B1 kinase. If comparing VacV-exposed samples, consider these sites when interpreting a phosphorylation-sensitive signal. Their listing does not establish a detectable shift on a standard blot.
Does this guide establish induction of RACK1?
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 RACK1?
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 M02145-1 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 RACK1 be quantified across cell fractions?
Quantitation · RACK1 is listed in the cytoplasm and as a peripheral cell-membrane protein, and it is a component of the small 40S ribosomal subunit. Compare the approximately 35 kDa band within consistently prepared fractions; a difference between fractions can reflect localization as well as total abundance.
Does RACK1's observed band match its predicted mass?
Interpretation · Yes. The observed band is approximately 35 kDa, close to the predicted 35.1 kDa for the 317-residue protein. The listed modifications alone do not establish a visible shift or explain any small difference in migration.
UniProt lists phosphotyrosine at position 52 by ABL1. For an ABL1-related comparison, a site-specific phospho-RACK1 measurement could address that site; a change in total RACK1 band intensity would not by itself establish phosphorylation at position 52.
RACK1 is listed as a monomer that also forms homodimers and homooligomers. That makes oligomerization relevant when assessing higher bands, but the feature does not identify any particular band. Confirm the band's identity before assigning it to an oligomer.
UniProt lists phosphotyrosine at position 52 by ABL1. For an ABL1-related comparison, a site-specific phospho-RACK1 measurement could address that site; a change in total RACK1 band intensity would not by itself establish phosphorylation at position 52.
RACK1 is listed as a monomer that also forms homodimers and homooligomers. That makes oligomerization relevant when assessing higher bands, but the feature does not identify any particular band. Confirm the band's identity before assigning it to an oligomer.
Boster reagents
RACK1 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-RACK1 Rabbit Monoclonal Antibody
Cat # M02145-1
The listed anti-RACK1 rabbit monoclonal antibody (M02145-1) has a Western blot image showing an approximately 35 kDa band in human cell, rat cell and liver, and mouse cell and liver lysates. Evidence is limited to this reported experiment.
Which to pick: M02145-1 is the only listed option. Its stated reactivity is human, mouse, and rat, and its WB image includes samples from all three. The caption reports a 1:500 primary dilution; use its sample types and conditions to guide your setup.
Source: BosterBio RACK1 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P63244.
- Human Protein Atlas. RACK1 tissue expression.
- PMC5642390 — target-verified WB comparison
- PMC3569494 — target-verified WB comparison
- PMC5233416 — target-verified WB comparison