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- Table of Contents
HSPA1B / Heat shock 70 kDa protein 1B · Western blot design guide
Design a Western Blot for HSPA1B
Real validated HSPA1B Western blot protocols, expected-band and isoform facts, troubleshooting for weak or shifted signal, and recommended anti-HSPA1B 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
HSPA1B 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 | ~70.1 kDa | |
| Observed band | ~70 kDa | |
| Gel | 5–20% (catalog PA1214) | |
| Positive control | Bronchus (IHC candidate; verify WB) +4 more | |
| Negative control | Bone marrow (IHC candidate; verify WB) |
Important caveats
Reasons your observed band may differ from the expected size.
| PTM | Phosphorylated + Acetylated | |
| Caveat | HSPA1A cross-reactivity | |
| Gene-set association | MSigDB C7 membership | |
| Isoform | 1 isoform(s) |
Section 1
Real Curated HSPA1B Western Blot Protocols
The PA1214 protocol combines labelled catalog values with standard starting conditions. Published comparisons retain their own sample, reagent and detection scope.
| Sample / lysate | rat brain, rat liver, rat spleen (catalog PA1214) |
| Gel % | 5–20% (catalog PA1214) |
| Load | 50ug; reducing conditions (catalog PA1214) |
| Transfer | a Nitrocellulose membrane at 150mA for 50-90 minutes (catalog PA1214) |
| Membrane | Nitrocellulose membrane (catalog PA1214) |
| Blocking | 5% Non-fat Milk/ TBS for 1.5 hour at RT (catalog PA1214) |
| Primary antibody | PA1214 · 0.5 μg/mL (catalog PA1214) |
| Primary incubation | overnight at 4°C (catalog PA1214) |
| Secondary antibody | goat anti-rabbit IgG-HRP, 1:10000 (catalog PA1214) |
| Secondary incubation | 1.5 hour at RT (catalog PA1214) |
| Wash | TBS-0.1%Tween 3 times with 5 minutes each (catalog PA1214) |
| Detection | ECL (catalog PA1214) |
Section 2
What Is the Expected HSPA1B Western Blot Band Size?
HSPA1B is predicted at 70.1 kDa and observed at ~70 kDa; its listed modifications do not establish a visible migration difference.
| Single band near 70 kDa | consistent with the 70.1 kDa prediction and the observed ~70 kDa band |
| Band near 70 kDa in whole-cell lysate | consistent with cytoplasmic localization |
| Band near 70 kDa despite acetylation | the listed acetylations have no demonstrated visible shift |
| Band near 70 kDa despite methylation | the listed methylations have no demonstrated visible shift |
💡Expected HSPA1B appearanceHSPA1B is predicted at 70.1 kDa and has an empirical band at ~70 kDa; confirm identity with antibody specificity and appropriate positive and negative controls.
| UniProt predicted mass | sets a 70.1 kDa reference for the observed ~70 kDa band |
| N-acetylalanine at residue 2 | has no demonstrated resolvable effect on migration |
| N6-acetyllysine at residues 77, 108, 246 and 348 | has no demonstrated resolvable effect on migration |
| Alternative lysine methylation at residue 561 | has no demonstrated resolvable effect on migration |
| Situation | Likely cause | Next action |
|---|---|---|
| No band in lysate | HSPA1B is cytoplasmic, so absence is unexplained by its listed localization | check lysate loading and a positive control |
| Band higher than expected | no listed glycosylation or disulfide feature establishes a higher band | compare with a positive control and verify antibody specificity |
| Band lower than expected | no listed signal peptide or propeptide explains a smaller mature form | check sample integrity and band identity |
| Multiple bands | the single listed isoform does not establish an isoform pattern | compare positive and negative controls and verify antibody specificity |
| Weak or no signal | HSPA1B detection may be limited by sample amount or antibody performance | check loading, transfer and a positive control |
Experiment evidence
Western blot images showing HSPA1B band
PA1214 · HSPA1B
← swipe / scroll for more images →
Real WB data behind the band-size expectations in this section.
Sample controls for HSPA1B Western blot
🧪HPA-IHC candidate guidance (verify in WB): For positive controls for HSPA1B in Western blot, you can use bronchus tissue, which HPA rates High.
Positive control: Bronchus (IHC candidate; verify WB)
Negative control: Bone marrow (IHC candidate; verify WB)
Loading controls: Run GAPDH, β-actin, and a total-protein stain alongside the samples.
⚠️Feasibility: HPA reports no detection in bone marrow, making a tissue negative feasible, though it should be confirmed by blot.
HPA tissue expression evidence for HSPA1B
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 |
|---|---|---|---|---|
| Bronchus | respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | neuronal cells | High | Protein (IHC) | HPA → |
| Cervix | glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | 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 |
|---|---|---|---|---|
| Bone marrow | hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | fibroblasts | Not detected | Protein (IHC) | HPA → |
| Spleen | cells in red pulp | Not detected | Protein (IHC) | HPA → |
| Adipose tissue | adipocytes | Low | Protein (IHC) | HPA → |
| Appendix | glandular cells | Low | Protein (IHC) | HPA → |
Section 3
Advanced HSPA1B Western Blot Tips
Deeper troubleshooting and optimisation questions for HSPA1B, answered from its protein features.
How should HSPA1B 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.
Do the supplied features support multiple HSPA1B isoforms?
Isoforms · Only one isoform is listed, with no alternative sequence. The supplied features therefore do not support assigning additional bands to HSPA1B isoforms.
Which HSPA1B modifications could matter when interpreting bands?
PTM · The features list acetylation at residues 2, 77, 108, 246 and 348; methylation at 469 and 561; and phosphorylation at 631, 633 and 636. These sites establish modification possibilities, but do not establish distinct visible bands.
Do not assign it from size alone. The supplied features list modifications but provide no evidence that any produces a separate visible band. They also list no alternative sequence, signal peptide or propeptide to explain another band.
Do not assign it from size alone. The supplied features list modifications but provide no evidence that any produces a separate visible band. They also list no alternative sequence, signal peptide or propeptide to explain another band.
Does this guide establish induction of HSPA1B?
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 HSPA1B?
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 PA1214 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.
Which band should be quantified for HSPA1B?
Quantitation · Start with the reported ~70 kDa band, which agrees with the 70.1 kDa predicted mass. Use the same band definition across samples; the features alone cannot establish that other bands represent HSPA1B.
Should HSPA1B run above its predicted mass?
Interpretation · Its predicted mass is 70.1 kDa, consistent with the reported ~70 kDa band. The listed modifications do not establish a visible shift or explain any difference between calculated and apparent mass.
Report residue 561 using the supplied UniProt numbering. It is listed with alternate dimethyllysine and trimethyllysine annotations; METTL21A is specified for trimethyllysine. Check the numbering convention before comparing it with antibody or paper descriptions.
HSPA1B carries a stress response keyword, but the supplied features specify no inducing condition or expected increase. Compare the ~70 kDa band across matched conditions and measure the response in the samples tested.
Report residue 561 using the supplied UniProt numbering. It is listed with alternate dimethyllysine and trimethyllysine annotations; METTL21A is specified for trimethyllysine. Check the numbering convention before comparing it with antibody or paper descriptions.
HSPA1B carries a stress response keyword, but the supplied features specify no inducing condition or expected increase. Compare the ~70 kDa band across matched conditions and measure the response in the samples tested.
Boster reagents
HSPA1B 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-Hsp70/HSPA1A/HSPA1B Antibody Picoband®
Cat # PA1214
PA1214 is an anti-Hsp70/HSPA1A/HSPA1B antibody listed for human, mouse, and rat. Its Western blot image shows an approximately 70 kDa band in rat and mouse tissue and cell lysates. The supplied evidence does not establish HSPA1B-specific detection.
Which to pick: PA1214 is the only listed option and has a Western blot image from rat and mouse samples. The caption reports 50 µg per lane and 0.5 µg/mL primary antibody. Human sample performance is not shown.
Source: BosterBio HSPA1B gene-info card — filtered to Western-blot-capable antibodies; each card shows that product's actual WB validation figure.
References
- UniProt Consortium. UniProt entry P0DMV9.
- Human Protein Atlas. HSPA1B tissue expression.
- PMC12808158 — target-verified WB comparison
- PMC6525452 — target-verified WB comparison
- PMC7465882 — target-verified WB comparison