Erk5 Antibody (Rabbit mAb) [P11L5]

Application: Reactivity: Mouse, Rat, Human

Lane 1: HeLa, Lane 2: NIH/3T3, Lane 3: PC-12

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.

Usage Information

Dilution
WB1:1000 IP1:30 IF1:100 FCM1:50

Application
WB, IP, IF, FCM

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
88 kDa 115 kDa, 88 kDa, 36 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	HeLa cells; A549 cells; NIH/3T3 cells; HAP1 cells; PC-12 cells
Negative Control

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: 5%. 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, 120 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 1:2000), 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: 5%. 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, 120 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 1:2000), 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.

IF
Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Permeabilization 1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes. (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.) Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

IF

Experimental Protocol:

Sample Preparation

1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.

2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.

3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.

4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.

Fixation

1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.

2. Wash the sample with PBS for 3 times, 3 minutes each time.

Permeabilization

1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.

(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)

Wash the sample with PBS for 3 times, 3 minutes each time.

Blocking

Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)

Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.

Immunofluorescence Staining (Day 1)

1. Remove the blocking solution and add the diluted primary antibody.

2. Incubate the sample in a humidified chamber at 4°C overnight.

Immunofluorescence Staining (Day 2)

1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.

2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.

3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.

4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.

5. Wash with PBST for 3 times, 5 minutes each time.

Mounting

1. Mount the sample with an anti-fade mounting medium.

2. Allow the slide to dry at room temperature overnight in the dark.

3. Store the slide in a slide storage box at 4°C, protected from light.

Datasheet & SDS

Biological Description

Specificity
Erk5 Antibody (Rabbit mAb) [P11L5] detects endogenous levels of total Erk5 protein.

Subcellular Location
Cytoplasm, Nucleus

Uniprot ID
Q13164

Clone
P11L5

Synonym(s)
BMK1, ERK5, PRKM7, MAPK7, Mitogen-activated protein kinase 7, MAP kinase 7, MAPK 7, Big MAP kinase 1, Extracellular signal-regulated kinase 5, BMK-1, ERK-5

Background
Erk5, also known as Big MAP Kinase 1 or BMK1 and encoded by the MAPK7 gene, belongs to the mitogen-activated protein kinase family alongside ERK1/2, the JNKs, and the p38 kinases, and functions as the terminal effector kinase of a three-tiered MAPK cascade built from the upstream kinases MEKK2 and MEKK3, the intermediate kinase MEK5, and ERK5 itself. ERK5 carries an N-terminal kinase domain sharing roughly half its sequence identity with ERK2, but distinguishes itself structurally from other MAPK family members through a large, unique C-terminal extension that contains a nuclear localization signal and a transcriptional activation domain, giving ERK5 a dual identity as both a catalytically active kinase and a direct transcriptional activator within the same molecule. Upon stimulation by mitogens or cellular stress, MEK5 phosphorylates the TEY motif within the ERK5 activation loop, activating the kinase domain, and this activation triggers autophosphorylation of multiple residues within the ERK5 C-terminus, which in turn drives translocation of ERK5 from the cytosol into the nucleus, where it interacts with and activates MEF2 family transcription factors, including MEF2D. Once in the nucleus, ERK5 activates transcription through two mechanistically distinct routes operating on the same substrate: direct phosphorylation of MEF2 by the ERK5 kinase domain, and a separate, kinase-independent mechanism in which the C-terminal transactivation domain itself functions as a transcriptional coactivator, meaning ERK5's transcriptional output does not depend solely on its catalytic activity. This transcriptional program drives expression of the Kruppel-like transcription factor genes LKLF/KLF2, and expression of a truncated ERK5 construct lacking the C-terminal transactivation domain fails to support LKLF expression in endothelial and T cells despite retaining kinase activity, directly demonstrating that the transactivation domain, not kinase activity alone, is required for this specific target gene. Because LKLF/KLF2 governs angiogenesis, T-cell quiescence, and vascular endothelial homeostasis, and because MEK5-ERK5 constitutes a unique, non-redundant signaling axis with no alternative upstream kinase, ERK5 loss in endothelial and T cells produces increased cell size and upregulated activation markers consistent with disrupted quiescence. The C-terminal domain is additionally subject to regulation by kinases other than ERK5 itself, including ERK1/2 and CDK1, which phosphorylate C-terminal residues independently of ERK5 catalytic activity, meaning ERK5-driven transcription integrates inputs from parallel signaling pathways beyond the canonical MEK5-ERK5 axis. Because pharmacological ERK5 kinase inhibitors can leave the C-terminal transactivation domain intact and, in some contexts, paradoxically increase ERK5-dependent transcription, both the kinase domain and the transactivation domain must be considered together when evaluating ERK5-targeted therapeutics.

Background

Erk5, also known as Big MAP Kinase 1 or BMK1 and encoded by the MAPK7 gene, belongs to the mitogen-activated protein kinase family alongside ERK1/2, the JNKs, and the p38 kinases, and functions as the terminal effector kinase of a three-tiered MAPK cascade built from the upstream kinases MEKK2 and MEKK3, the intermediate kinase MEK5, and ERK5 itself. ERK5 carries an N-terminal kinase domain sharing roughly half its sequence identity with ERK2, but distinguishes itself structurally from other MAPK family members through a large, unique C-terminal extension that contains a nuclear localization signal and a transcriptional activation domain, giving ERK5 a dual identity as both a catalytically active kinase and a direct transcriptional activator within the same molecule. Upon stimulation by mitogens or cellular stress, MEK5 phosphorylates the TEY motif within the ERK5 activation loop, activating the kinase domain, and this activation triggers autophosphorylation of multiple residues within the ERK5 C-terminus, which in turn drives translocation of ERK5 from the cytosol into the nucleus, where it interacts with and activates MEF2 family transcription factors, including MEF2D. Once in the nucleus, ERK5 activates transcription through two mechanistically distinct routes operating on the same substrate: direct phosphorylation of MEF2 by the ERK5 kinase domain, and a separate, kinase-independent mechanism in which the C-terminal transactivation domain itself functions as a transcriptional coactivator, meaning ERK5's transcriptional output does not depend solely on its catalytic activity. This transcriptional program drives expression of the Kruppel-like transcription factor genes LKLF/KLF2, and expression of a truncated ERK5 construct lacking the C-terminal transactivation domain fails to support LKLF expression in endothelial and T cells despite retaining kinase activity, directly demonstrating that the transactivation domain, not kinase activity alone, is required for this specific target gene. Because LKLF/KLF2 governs angiogenesis, T-cell quiescence, and vascular endothelial homeostasis, and because MEK5-ERK5 constitutes a unique, non-redundant signaling axis with no alternative upstream kinase, ERK5 loss in endothelial and T cells produces increased cell size and upregulated activation markers consistent with disrupted quiescence. The C-terminal domain is additionally subject to regulation by kinases other than ERK5 itself, including ERK1/2 and CDK1, which phosphorylate C-terminal residues independently of ERK5 catalytic activity, meaning ERK5-driven transcription integrates inputs from parallel signaling pathways beyond the canonical MEK5-ERK5 axis. Because pharmacological ERK5 kinase inhibitors can leave the C-terminal transactivation domain intact and, in some contexts, paradoxically increase ERK5-dependent transcription, both the kinase domain and the transactivation domain must be considered together when evaluating ERK5-targeted therapeutics.

References
https://pubmed.ncbi.nlm.nih.gov/16166637/ https://pubmed.ncbi.nlm.nih.gov/32170057/

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

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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%