Cyclophilin B Antibody (Rabbit mAb) [F10F4]

Application: Reactivity: Mouse, Rat, Human

Lane 1: HepG2, Lane 2: Hela, Lane 3: A431, Lane 4: PC12

Experiment Essentials

WB
Recommended wet transfer conditions: 200 mA, 60 min.

Usage Information

Dilution
WB1:1000 - 1:10000

Application
WB

Reactivity
Mouse, Rat, Human

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
24 kDa 18 kDa, 24 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	Mouse heart tissue; Rat heart tissue; HAP1 cells; U87-MG cells; HepG2 cells; HeLa cells; A431 cells; NIH/3T3 cells; PC-12 cells
Negative Control	Jurkat cells

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 60 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 60 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Datasheet & SDS

Biological Description

Specificity
Cyclophilin B Antibody (Rabbit mAb) [F10F4] detects endogenous levels of total Cyclophilin B protein.

Subcellular Location
Endoplasmic reticulum, Mitochondrion

Uniprot ID
P23284

Clone
F10F4

Synonym(s)
B \| Cyclophilin B \| cyclophilin-like protein \| CYP-S1 \| CYPB \| epididymis secretory protein Li 39 \| HEL-S-39 \| MGC14109 \| MGC2224 \| OI9 \| Peptidyl-prolyl cis-trans isomerase B \| peptidylprolyl isomerase B \| peptidylprolyl isomerase B (cyclophilin B) \| PPIase B \| PPIB \| Rotamase B \| S-cyclophilin \| SCYLP

Background
Cyclophilin B is a secretory pathway peptidyl‑prolyl cis‑trans isomerase of the cyclophilin family that functions as a molecular chaperone in the endoplasmic reticulum and participates in extracellular signaling after secretion. The protein carries an N‑terminal signal sequence that directs co‑translational translocation into the endoplasmic reticulum lumen, where it accumulates and assists protein folding by catalyzing cis‑trans isomerization of proline imidic peptide bonds in nascent and resident polypeptides, with cyclosporin A binding altering its trafficking through the secretory pathway. ER‑localized cyclophilin B shows a distribution between the endoplasmic reticulum and later secretory compartments, and cyclosporin A association leads to retention changes that affect its movement toward the Golgi and extracellular space, indicating that drug interaction modulates access of cyclophilin B to client proteins and secreted cargo. The secreted fraction engages cell surface receptors and matrix components; cyclophilin B acts as a ligand for the CD147 receptor and interacts with heparan sulfate proteoglycans on T lymphocytes, granulocytes and macrophages, which enables chemotaxis, adhesion of T cells to fibronectin and regulation of downstream events such as mitogen‑activated protein kinase activation, calcium transport and expression of the pro‑apoptotic protein Bim. This combination of ER chaperone activity and extracellular receptor binding places cyclophilin B in pathways that connect protein folding homeostasis with inflammatory signaling and cell migration, and its secretion in response to inflammatory stimuli and oxidative stress contributes to tissue or systemic inflammation without directly inducing pro‑inflammatory cytokine production on its own. Structural features include the conserved cyclophilin PPIase domain that provides the active site for proline isomerization, together with regions that support interaction with CD147 and heparan sulfate proteoglycans at the cell surface, allowing the same polypeptide to operate in folding and signaling environments. In adipogenesis and bone cell biology, cyclophilin B has been described as a molecular chaperone that promotes differentiation through modulation of signaling cascades such as AKT/mTOR or JAK2/STAT3, linking its PPIase activity and receptor interactions to transcriptional control of lineage‑specific genes in preadipocytes and osteoblast‑like cells. Serum and tissue levels of cyclophilin B increase under inflammatory and metabolic stress, including metabolic syndrome, where constitutive secretion correlates with prevalence and severity and supports a role in metabolic inflammation through its participation in cell–cell communication and leukocyte trafficking rather than direct cytokine induction. In viral infection, cyclophilin B functions as a host factor for hepatitis C virus replication by binding the viral RNA polymerase NS5B, stimulating its RNA binding activity and supporting efficient genome replication, and RNA interference‑mediated reduction of cyclophilin B or disruption of NS5B–cyclophilin B interaction lowers replication efficiency, establishing this PPIase as a regulator within the HCV replication machinery. Cyclophilin B expression is increased in several tumor types and inflammatory diseases such as rheumatoid arthritis and psoriasis, and its extracellular activity through CD147 and heparan sulfate proteoglycans contributes to chemotaxis, adhesion and signaling changes in immune cells, situating it at a junction of protein folding quality control, viral replication, metabolic and inflammatory signaling, and tumor progression that is accessible both to genetic manipulation and pharmacologic inhibition targeting cyclophilin–ligand interactions.

Background

Cyclophilin B is a secretory pathway peptidyl‑prolyl cis‑trans isomerase of the cyclophilin family that functions as a molecular chaperone in the endoplasmic reticulum and participates in extracellular signaling after secretion. The protein carries an N‑terminal signal sequence that directs co‑translational translocation into the endoplasmic reticulum lumen, where it accumulates and assists protein folding by catalyzing cis‑trans isomerization of proline imidic peptide bonds in nascent and resident polypeptides, with cyclosporin A binding altering its trafficking through the secretory pathway. ER‑localized cyclophilin B shows a distribution between the endoplasmic reticulum and later secretory compartments, and cyclosporin A association leads to retention changes that affect its movement toward the Golgi and extracellular space, indicating that drug interaction modulates access of cyclophilin B to client proteins and secreted cargo. The secreted fraction engages cell surface receptors and matrix components; cyclophilin B acts as a ligand for the CD147 receptor and interacts with heparan sulfate proteoglycans on T lymphocytes, granulocytes and macrophages, which enables chemotaxis, adhesion of T cells to fibronectin and regulation of downstream events such as mitogen‑activated protein kinase activation, calcium transport and expression of the pro‑apoptotic protein Bim. This combination of ER chaperone activity and extracellular receptor binding places cyclophilin B in pathways that connect protein folding homeostasis with inflammatory signaling and cell migration, and its secretion in response to inflammatory stimuli and oxidative stress contributes to tissue or systemic inflammation without directly inducing pro‑inflammatory cytokine production on its own. Structural features include the conserved cyclophilin PPIase domain that provides the active site for proline isomerization, together with regions that support interaction with CD147 and heparan sulfate proteoglycans at the cell surface, allowing the same polypeptide to operate in folding and signaling environments. In adipogenesis and bone cell biology, cyclophilin B has been described as a molecular chaperone that promotes differentiation through modulation of signaling cascades such as AKT/mTOR or JAK2/STAT3, linking its PPIase activity and receptor interactions to transcriptional control of lineage‑specific genes in preadipocytes and osteoblast‑like cells. Serum and tissue levels of cyclophilin B increase under inflammatory and metabolic stress, including metabolic syndrome, where constitutive secretion correlates with prevalence and severity and supports a role in metabolic inflammation through its participation in cell–cell communication and leukocyte trafficking rather than direct cytokine induction. In viral infection, cyclophilin B functions as a host factor for hepatitis C virus replication by binding the viral RNA polymerase NS5B, stimulating its RNA binding activity and supporting efficient genome replication, and RNA interference‑mediated reduction of cyclophilin B or disruption of NS5B–cyclophilin B interaction lowers replication efficiency, establishing this PPIase as a regulator within the HCV replication machinery. Cyclophilin B expression is increased in several tumor types and inflammatory diseases such as rheumatoid arthritis and psoriasis, and its extracellular activity through CD147 and heparan sulfate proteoglycans contributes to chemotaxis, adhesion and signaling changes in immune cells, situating it at a junction of protein folding quality control, viral replication, metabolic and inflammatory signaling, and tumor progression that is accessible both to genetic manipulation and pharmacologic inhibition targeting cyclophilin–ligand interactions.

References
https://pubmed.ncbi.nlm.nih.gov/7909608/ https://pubmed.ncbi.nlm.nih.gov/15989969/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

Tech Support

Handling Instructions

Tel: +1-832-582-8158 Ext:3

If you have any other enquiries, please leave a message.

Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%