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- Table of Contents
GRB2 / Growth factor receptor-bound protein 2 · Western blot design guide
Design a Western Blot for GRB2
Real validated GRB2 Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-GRB2 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
GRB2 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 | ~25.2 kDa | |
| Observed band | Approximately 25 kDa | |
| Gel | 5–20% (catalog A00351-1) | |
| Positive control | Appendix (IHC candidate; verify WB) +4 more | |
| Negative control | Heart muscle (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 | 2 isoform(s) |
Section 1
Real Curated GRB2 Western Blot Protocols
The A00351-1 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | human 293T, human THP-1, human HL-60 (catalog A00351-1) |
| Gel % | 5–20% (catalog A00351-1) |
| Load | 30 ug; reducing conditions (catalog A00351-1) |
| Transfer | a nitrocellulose membrane at 150 mA for 50-90 minutes (catalog A00351-1) |
| Membrane | nitrocellulose membrane (catalog A00351-1) |
| Blocking | 5% non-fat milk/TBS for 1.5 hour at RT (catalog A00351-1) |
| Primary antibody | A00351-1 · 0.5 μg/mL (catalog A00351-1) |
| Primary incubation | overnight at 4°C (catalog A00351-1) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:5000 (catalog A00351-1) |
| Secondary incubation | 1.5 hour at RT (catalog A00351-1) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog A00351-1) |
| Detection | ECL (catalog A00351-1) |
Section 2
What Is the Expected GRB2 Western Blot Band Size?
GRB2 is predicted at 25.2 kDa and observed at approximately 25 kDa; no cause for the small difference is established.
| Band at approximately 25 kDa | Matches the empirical GRB2 band in reducing whole-cell lysates. |
| Band near 50 kDa under non-reducing conditions | Could reflect the reported GRB2 homodimer if it survives electrophoresis. |
| Additional bands at different positions | Could represent isoforms 1 and 2 if their migration differs; band identity needs confirmation. |
| Band near 25 kDa in a nuclear fraction | Consistent with GRB2's reported nuclear localization. |
💡Expected GRB2 appearanceGRB2 has a predicted mass of 25.2 kDa and an empirical band at approximately 25 kDa in reducing whole-cell lysates; confirm band identity with an appropriate control.
| Predicted GRB2 mass | Places the full-length protein near 25.2 kDa. |
| GRB2 homodimer | Could appear near twice the monomer mass if preserved under non-reducing conditions. |
| Isoform 1 | Its size relative to isoform 2 is not supplied. |
| Isoform 2 | May differ in mass from isoform 1, but its size and migration are not supplied. |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | Recovery of GRB2 from its cellular compartments may be inadequate. | Check extraction and loading with a positive lysate control. |
| Band higher than expected | The reported homodimer may persist if reduction is incomplete. | Compare reducing and non-reducing samples. |
| Band lower than expected | An alternative isoform is possible, but its migration is unknown. | Confirm identity with an independent antibody or isoform-specific assay. |
| Multiple bands | Isoforms 1 and 2 could migrate differently, though distinct bands are not established. | Compare isoform-specific expression or use an independent antibody. |
| Weak or no signal | GRB2 recovery from the sampled compartment may be low. | Check fraction recovery, loading, and a positive lysate control. |
Experiment evidence
Western blot images showing GRB2 band
A00351-1 · GRB2
A00351 · GRB2
M00351 · GRB2
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for GRB2 Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for GRB2 in Western blot, you can use appendix lysate.
Positive control: Appendix (IHC candidate; verify WB)
Negative control: Heart muscle (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: HPA reports high expression in appendix and no detection in heart muscle, supporting tissue controls.
HPA tissue expression evidence for GRB2
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 | lymphoid tissue | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Lymph node | germinal center cells | High | Protein (IHC) | HPA → |
| Placenta | decidual cells | High | Protein (IHC) | HPA → |
| Tonsil | germinal center cells | High | Protein (IHC) | HPA → |
Lower expression tissues · IHC evidence, not confirmed WB-negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Heart muscle | cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | glandular cells | Low | Protein (IHC) | HPA → |
| Bronchus | respiratory epithelial cells | Low | Protein (IHC) | HPA → |
| Cervix | glandular cells | Low | Protein (IHC) | HPA → |
| Esophagus | squamous epithelial cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced GRB2 Western Blot Tips
Deeper troubleshooting and optimisation questions for GRB2, answered from its protein features.
Where should the main GRB2 band appear?
Band shift · GRB2 has a predicted mass of 25.2 kDa, consistent with the reported band near 25 kDa. Use that position as a guide when identifying the band.
Could GRB2 isoforms produce bands at different positions?
Isoforms · UniProt lists isoforms 1 and 2. Isoform 2 lacks residues 60–100 of the canonical sequence, so it is expected to be smaller. Whether both appear depends on their presence in the sample and antibody recognition.
Do GRB2 modifications explain a shifted band?
PTM · UniProt lists N-acetylmethionine at residue 1, N6-acetyllysine at 6, 50 and 109, phosphotyrosine at 209, and phosphothreonine at 211. These are UniProt coordinates; antibody or paper numbering may differ. Their presence alone does not establish a visible shift or explain an apparent mass difference.
Is growth factor stimulation relevant to GRB2 analysis?
Induction · GRB2 associates through its SH2 domain with activated, tyrosine-phosphorylated EGF and PDGF receptors. Stimulation of those pathways may therefore be relevant when comparing GRB2-associated signaling, but the supplied features do not establish increased GRB2 abundance or phosphorylation after stimulation.
How should transfer be checked for GRB2?
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 A00351-1 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 GRB2 bands be quantified across fractions?
Quantitation · GRB2 is listed in the cytoplasm, nucleus and endosome, with Golgi localization also annotated. Compare like fractions across samples and identify which band or isoform is being measured before interpreting a change as a change in total GRB2.
How should unexpected GRB2 bands be interpreted?
Interpretation · Consider the smaller isoform 2, which lacks canonical residues 60–100, and the listed modifications when evaluating band identity. GRB2 is also annotated as a homodimer, but that annotation alone does not predict an extra band on a Western blot. Band position alone cannot establish identity.
Boster reagents
GRB2 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-GRB2 Antibody Picoband®
Cat # A00351-1
Real WB data
Anti-GRB2 Antibody (Y209)
Cat # A00351
Real WB data
Anti-GRB2 Monoclonal Antibody
Cat # M00351
All three listed anti-GRB2 antibodies have Western blot images. A00351-1 shows an approximately 25 kDa band in human 293T, THP-1, and HL-60 lysates; A00351 uses Ramos lysate, and M00351 uses HEK293 lysate. The supplied evidence does not establish performance beyond these examples.
Which to pick: For human samples, choose based on the closest documented lysate and conditions: A00351-1 for 293T, THP-1, or HL-60; A00351 for Ramos; M00351 for HEK293. Only M00351 lists mouse and rat reactivity, although its supplied WB image uses HEK293.
Source: BosterBio GRB2 gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P62993.
- Human Protein Atlas. GRB2 tissue expression.
- PMC4191928 — target-verified WB comparison
- PMC6116178 — target-verified WB comparison
- PMC3792889 — target-verified WB comparison