Home Primary Antibodies eIF3B Rabbit mAb

eIF3B Rabbit mAb

Catalog No.: F3620

    Application: Reactivity:

    Experiment Essentials

    WB
    Recommended WB dilution ratio: 1:10000

    Usage Information

    Dilution
    1:10000-1:50000
    1:500
    1:10 - 1:100
    Application
    WB, IF, FCM
    Source
    Rabbit
    Reactivity
    Human
    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
    92 kDa 116 kDa
    *Why do the predicted and actual molecular weights differ?
    The following reasons may explain differences between the predicted and actual protein molecular weight.

    Exprimental Methods

    WB
    Experimental Protocol:
     
    Sample preparation
    1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Tris-Triton Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature.
    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/Tris-Triton Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.
    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/Tris-Triton Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.
    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 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:10000), 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：
     
    Specimen Preparation 
    1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% Paraformaldehyde diluted in 1X PBS.
    NOTE: Paraformaldehyde is toxic, use only in a fume hood.
    2. Fix cells for 15 min at room temperature.
    3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
    4. Proceed with Immunostaining.
     
    Immunostaining
    1. Add theblocking buffer and incubate for 60 min at RT.
    2. Prepare primary antibody diluent in antibody dilution buffer as recommended .
    3. Aspirate blocking solution, apply diluted primary antibody.
    4. Incubate overnight at 4°C.
    5. Rinse three times in 1X PBS for 5 min each.
    6. Incubate specimens in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
    7. Rinse three times in 1X PBS for 5 min each.
    8. Mount slides usingmounting medium with DAPI and cover with coverslips.
    9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 38°C protected from light.
     

    Datasheet & SDS

    Biological Description

    Specificity

    eIF3B Rabbit mAb recognizes endogenous levels of total eIF3B protein.
    Synonym(s)
    EIF3S9, Eukaryotic translation initiation factor 3 subunit B, Eukaryotic translation initiation factor 3 subunit 9, Prt1 homolog, eIF-3-eta, eIF3 p110, eIF3 p116, hPrt1
    Uniprot ID
    P55884
    Clone
    K11L7
    Background

    eIF3B (Translation initiation factor 3b) is a core subunit of the eukaryotic translation initiation factor 3 (eIF3) complex and serves as a crucial scaffolding protein essential for accurate translation initiation. Structurally, eIF3b contains an N-terminal RNA recognition motif (RRM) and a WD40β domain, and in yeast, an additional C-terminal domain (CTD) that mediates interactions with other eIF3 subunits like eIF3g and eIF3i. It is ubiquitously expressed in human tissues and upregulated in multiple cancers, including gastric, bladder, and prostate, where it promotes tumor growth and is linked to poor prognosis. Functionally, eIF3b is critical for ribosomal 40S subunit recruitment, start codon scanning, and the assembly of the pre-initiation complex, and its depletion disrupts global protein synthesis, as shown by reduced polysome formation. Despite its universal role in translation, eIF3b appears to preferentially enhance the translation of proto-oncogenes while repressing tumor suppressor mRNAs, underscoring its emerging role as an oncogene and highlighting the need for further investigation into its regulatory mechanisms.
    References

    Tech Support

    Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

    Handling Instructions

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