E2A Antibody (Rat mAb) [K20N18] | Primary Antibodies

Application: Reactivity: Rat, Mouse

Lane 1: NIH/3T3, Lane 2: RAW264.7

Experiment Essentials

This antibody requires an anti-rat secondary antibody.

Usage Information

Dilution
WB1:1000 IHC1:200

Application
WB, IHC

Reactivity
Rat, Mouse

Source
Rat 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
68 kDa 67 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 spleen tissue; Rat spleen tissue; C2C12 cells; WEHI-231 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/Nuclear 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/Nuclear 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/Nuclear 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, 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 1:1000), 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/Nuclear 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/Nuclear 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/Nuclear 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, 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 1:1000), 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.

IHC
Experimental Protocol： Deparaffinization/Rehydration 1. Deparaffinize/hydrate sections: 2. Incubate sections in three washes of xylene for 5 min each. 3. Incubate sections in two washes of 100% ethanol for 10 min each. 4. Incubate sections in two washes of 95% ethanol for 10 min each. 5. Wash sections two times in dH2O for 5 min each. 6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min. Staining 1. Wash sections in dH2O three times for 5 min each. 2. Incubate sections in 3% hydrogen peroxide for 10 min. 3. Wash sections in dH2O two times for 5 min each. 4. Wash sections in wash buffer for 5 min. 5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature. 6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C. 7. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature. 9. Wash sections three times with wash buffer for 5 min each. 10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use. 11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity. 12. Immerse slides in dH2O. 13. If desired, counterstain sections with hematoxylin. 14. Wash sections in dH2O two times for 5 min each. 15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each. 16. Mount sections with coverslips and mounting medium.

IHC

Experimental Protocol：

Deparaffinization/Rehydration

1. Deparaffinize/hydrate sections:

2. Incubate sections in three washes of xylene for 5 min each.

3. Incubate sections in two washes of 100% ethanol for 10 min each.

4. Incubate sections in two washes of 95% ethanol for 10 min each.

5. Wash sections two times in dH2O for 5 min each.

6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.

Staining

1. Wash sections in dH2O three times for 5 min each.

2. Incubate sections in 3% hydrogen peroxide for 10 min.

3. Wash sections in dH2O two times for 5 min each.

4. Wash sections in wash buffer for 5 min.

5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.

6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.

7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.

8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.

9. Wash sections three times with wash buffer for 5 min each.

10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.

11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.

12. Immerse slides in dH2O.

13. If desired, counterstain sections with hematoxylin.

14. Wash sections in dH2O two times for 5 min each.

15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.

16. Mount sections with coverslips and mounting medium.

Datasheet & SDS

Biological Description

Specificity
E2A Antibody (Rat mAb) [K20N18] detects endogenous levels of total E2A protein.

Subcellular Location
Nucleus

Uniprot ID
P15806

Clone
K20N18

Synonym(s)
Alf2, Me2, Tcfe2a, Tcf3, Transcription factor E2-alpha, Immunoglobulin enhancer-binding factor E12/E47, Transcription factor 3, Transcription factor A1, TCF-3

Background
TCF3, also known as E2A, is a basic helix–loop–helix (bHLH) transcription factor of the E‑protein family that generates the E12 and E47 isoforms by alternative splicing and controls lineage-specific gene expression by binding E‑box motifs 5′‑CANNTG‑3′ in enhancer and promoter regions of diverse developmental and immune genes. The protein contains N‑terminal transactivation domains that engage coactivators such as CBP/p300, a central bHLH dimerization and DNA-binding module that forms homodimers or heterodimers with tissue‑restricted bHLH partners, and regulatory regions that interact with inhibitor of DNA-binding (ID) proteins and ubiquitin–ligase adaptors, allowing dynamic modulation of its activity through protein–protein interactions and Notch-induced ubiquitination and proteasomal degradation. E2A dimers bind canonical E‑boxes in immunoglobulin gene enhancers (for example the κ‑E2 site in the κ light chain enhancer) and insulin gene regulatory elements, and cooperate with B‑lineage transcription factors such as EBF1 and PAX5 to activate networks required for B‑cell lineage commitment, early B lymphopoiesis, germinal center B‑cell differentiation and plasma cell development, with gene‑dosage studies showing that two intact TCF3 alleles are needed to sustain normal B‑cell numbers, class‑switching and immunoglobulin secretion. In T‑lineage development, E2A drives expression of recombination-activating genes (RAG), promotes survival of precursor and mature lymphocytes, and directly enhances Hes1 transcription as a Notch pathway target, integrating bHLH-driven E‑box binding with canonical Notch signaling to influence early lymphoid progenitor differentiation and central nervous system development. Heterodimers between TCF3 and tissue‑specific bHLH factors, including NEUROD1 and ATOH7, facilitate binding to neuronal consensus sites such as 5′‑CAGGTG‑3′ and positively regulate transcriptional programs for neuronal differentiation and mesenchymal–epithelial transition, so E2A serves as a broadly used cofactor that steers cell‑fate decisions in neural and mesenchymal lineages by partnering with lineage‑restricted bHLH proteins. Germline loss‑of‑function or dominant‑negative TCF3 mutations cause severe agammaglobulinemia with early blocks in B‑cell development and combined defects in B‑cell numbers, memory formation and plasmablast differentiation, and recent human and murine studies define a haploinsufficiency state in which monoallelic TCF3 mutations reduce wild-type protein expression, produce B‑cell and serum immunoglobulin abnormalities and dysregulated lymphocyte transcriptomes while showing incomplete clinical penetrance. Somatic chromosomal translocations involving TCF3 generate oncogenic fusion proteins in lymphoid malignancies: t(1;19) produces E2A–PBX1, t(17;19) generates E2A–HLF, and additional fusions with TFPT and ZNF384 occur in acute leukemias; these chimeric proteins aberrantly recruit E2A transactivation and bHLH DNA-binding modules to ectopic target genes, driving pre‑B acute lymphoblastic leukemia and related lymphoid cancers. Genome-wide association data identify intronic TCF3 variants at 19p13.3 as a Hodgkin lymphoma susceptibility locus, with the minor allele enhancing ZBTB7A binding, increasing TCF3 expression and associating with reduced disease risk, supporting a tumor‑suppressor role for E2A through maintenance of the B‑cell phenotype in Hodgkin lymphomagenesis.

Background

TCF3, also known as E2A, is a basic helix–loop–helix (bHLH) transcription factor of the E‑protein family that generates the E12 and E47 isoforms by alternative splicing and controls lineage-specific gene expression by binding E‑box motifs 5′‑CANNTG‑3′ in enhancer and promoter regions of diverse developmental and immune genes. The protein contains N‑terminal transactivation domains that engage coactivators such as CBP/p300, a central bHLH dimerization and DNA-binding module that forms homodimers or heterodimers with tissue‑restricted bHLH partners, and regulatory regions that interact with inhibitor of DNA-binding (ID) proteins and ubiquitin–ligase adaptors, allowing dynamic modulation of its activity through protein–protein interactions and Notch-induced ubiquitination and proteasomal degradation. E2A dimers bind canonical E‑boxes in immunoglobulin gene enhancers (for example the κ‑E2 site in the κ light chain enhancer) and insulin gene regulatory elements, and cooperate with B‑lineage transcription factors such as EBF1 and PAX5 to activate networks required for B‑cell lineage commitment, early B lymphopoiesis, germinal center B‑cell differentiation and plasma cell development, with gene‑dosage studies showing that two intact TCF3 alleles are needed to sustain normal B‑cell numbers, class‑switching and immunoglobulin secretion. In T‑lineage development, E2A drives expression of recombination-activating genes (RAG), promotes survival of precursor and mature lymphocytes, and directly enhances Hes1 transcription as a Notch pathway target, integrating bHLH-driven E‑box binding with canonical Notch signaling to influence early lymphoid progenitor differentiation and central nervous system development. Heterodimers between TCF3 and tissue‑specific bHLH factors, including NEUROD1 and ATOH7, facilitate binding to neuronal consensus sites such as 5′‑CAGGTG‑3′ and positively regulate transcriptional programs for neuronal differentiation and mesenchymal–epithelial transition, so E2A serves as a broadly used cofactor that steers cell‑fate decisions in neural and mesenchymal lineages by partnering with lineage‑restricted bHLH proteins. Germline loss‑of‑function or dominant‑negative TCF3 mutations cause severe agammaglobulinemia with early blocks in B‑cell development and combined defects in B‑cell numbers, memory formation and plasmablast differentiation, and recent human and murine studies define a haploinsufficiency state in which monoallelic TCF3 mutations reduce wild-type protein expression, produce B‑cell and serum immunoglobulin abnormalities and dysregulated lymphocyte transcriptomes while showing incomplete clinical penetrance. Somatic chromosomal translocations involving TCF3 generate oncogenic fusion proteins in lymphoid malignancies: t(1;19) produces E2A–PBX1, t(17;19) generates E2A–HLF, and additional fusions with TFPT and ZNF384 occur in acute leukemias; these chimeric proteins aberrantly recruit E2A transactivation and bHLH DNA-binding modules to ectopic target genes, driving pre‑B acute lymphoblastic leukemia and related lymphoid cancers. Genome-wide association data identify intronic TCF3 variants at 19p13.3 as a Hodgkin lymphoma susceptibility locus, with the minor allele enhancing ZBTB7A binding, increasing TCF3 expression and associating with reduced disease risk, supporting a tumor‑suppressor role for E2A through maintenance of the B‑cell phenotype in Hodgkin lymphomagenesis.

References
https://pubmed.ncbi.nlm.nih.gov/27166193/ https://pubmed.ncbi.nlm.nih.gov/27261530/

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%