HUABIO is dedicated to developing high-quality antibodies that advance innovation. We're passionate about the accuracy, efficiency, and consistency of our products. That's why we've invested in new production platforms, like recombinant rabbit monoclonals, alpaca nanobodies, and adopted aggressive QA standards to deliver cutting-edge antibodies with uncompromised quality. HUABIO is located in the Greater Boston area, United States


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ET1611-58 ACE-2 (SN0754) ET1607-2 CD63 (SY21-02)
ET1609-69 Neuropilin-1 (ST05-30) ET1610-66 HLA DR/DP (SC06-78)
ET1611-87 CD81 (SN206-01) ET1702-61 HE4/WFDC2 (JF62-09)
ET1601-9 CD9 (SA35-08) ET1702-85 GLI-1 (JF09-08)
ET1610-93 NLRP3/NALP3 (SC06-23) ET1702-86 MITF (JF100-01)
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ET1611-65 CCR2 (SN707) ET1704-16 Choline Acetyltransferase/ChAT (JA67-11)
ET1606-49 Integrin beta 3/CD61 (SJ19-09) ET1704-69 MMP-9 (JA80-73)
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ET1705-51 MLKL [p Ser345] (JM92-37) ET1606-48 MMP-14/MT1-MMP (SJ18-09)
ET1612-37 ATF4 (SD20-92) EM1701-26 PCSK9 [2F1]
ET1610-85 CD14 (SC69-02) ET1601-18 VCAM-1/CD106 (SA05-04)
1007-8 CD80/B7-1 ET1605-41 V5tag
ET1602-22 CCR7 (SR36-04) ET1610-89 IRF7 (SC0617)
ET1703-46 Hemoglobin gamma A (JM84-10) ET1703-16 NG2/MCSP (JM10-13)
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ET1704-59 Glut3 (JA50-31) ER1706-27 PINK1
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R1411-1 LINE-1 ORF1p ET1610-42 Cytokeratin 14 (SC65-06)
ET1604-28 VEGF ET1612-91 Collagen VI alpha 1 (SD83-03)
ET1610-94 GAP43 ET1701-82 MKP-1/DUSP1 (JJ0930)
ET1704-44 Leptin R (JA73-01) ET1703-27 S1P1/EDG-1 (JM10-66)
ET1602-42 MMP-12 (SR03-23) ET1705-6 Angiopoietin 2
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ET1603-14 eIF2A [p Ser51] (SZ01-06) ET1702-74 CD19 (JF099-9)
ET1611-57 Integrin alpha 2/CD49b (SN0752) ET1702-99 Complement C3a (JF10-30)
ET1702-29 LYVE-1 (JF0979) R1706-20 MRP8/S100A8
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ER50102 MuERVL-Gag ET1606-42 BDNF (SJ12-09)
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ET1703-42 Syndecan-1/CD138 (JM11-21) ET1612-13 S100A4 (SD200-08)
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ET1703-17 IFN-gamma (JM10-10) ET1705-15 PMP22 (JM52-30)
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ET1601-10 Glucose Transporter GLUT1 ET1606-31 CD8 (SI18-01)
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ET1608-32 Cytokeratin 18 (SU0338) ET1611-40 A20/TNFAIP3 (SN07-31)
ET1612-14 IRF3 (SD2062) ET1701-38 SOX1 (JJ20-40)
ET1606-47 LDL R (SJ0197) ET1702-47 MHC Class I (JF10-38)
ET1702-49 PDGF R alpha (JF104-6)

Protocols

Western Blot Introduction

Western Blotting is an analytical Immunoblotting Technique to detect specific proteins in a cell extract or tissue homogenate. Western Blotting relies on the specific binding between the protein-of-interest and an antibody raised against this particular protein.

Step-by-step western blot protocol

All steps are carried out at room temperature unless otherwise indicated. Recipes for all standardized solutions highlighted in bold can be found on the internet or the back of a textbook.

SDS PAGE

  • Construct an SDS-PAGE gel according to the molecular weight (MW) of your target protein(s). (Recommendations and gel recipes are presented at the end of this protocol)
  • Tip 1: Tris-tricine gels separate low MW proteins (<20 kDa) better than Tris-glycine gels.
  • Prepare samples in microfuge tubes. Add 4X SDS sample buffer so the total protein amount is 30–50 μg per sample (according to the protein amount measured by Bradford or BCA protein assay).
  • Flick microfuge tubes to mix samples, and then heat to 95-100°C for 5 minutes.
  • Set up electrophoresis apparatus and immerse in a 1x running buffer. Remove gel combs and cleanse wells of any residual stacking gel.
  • Load samples and protein markers onto the gel using gel loading tips. Set electrophoresis power pack to 80V (through the stacking gel), before increasing it to 120V when the protein front reaches the separation gel.

Protein transfer

  • PVDF membranes (or PSQ membranes with 0.22 μm micropores when MW of the target is <30 kDa) are strongly recommended. Soak membranes in methanol for 30 seconds before moving to transfer buffer. Soak the filter papers and sponges in the transfer buffer as well.
  • Sequentially assemble the transfer constituents according to the illustration on page 7 of this booklet and ensure no bubbles lie between any of the layers. Apply semi-dry or wet transfer systems according to the manufacturer’s instructions.

Immunoblotting

  • After transfer, wash the membrane twice with distilled water, and using a pencil, mark bands of the MW ladder on the membrane. If desired, stain the membrane with commercial Ponceau red solution for 1 min to visualize protein bands, then wash any Ponceau red staining with copious amounts of 1x TBST.
  • Block with 1x TBST containing (2-5%) nonfat dry milk (or 1-5% BSA for the detection of phospho-epitope antibodies) with constant rocking for 1 hour or overnight at 4°C.
  • Dilute primary antibody in blocking solution with a starting dilution ratio of 1:1000. (Optimal dilutions should be determined experimentally.) Incubate the membrane with primary antibody for 1 hour at room temperature, or overnight at 4°C.
  • Wash membrane three times with 1x TBST for 10 minutes each.
  • Incubate the membrane with a suitable HRP-conjugated secondary antibody (recognizing the host species of the primary antibody), diluted at 1:5000–1:50000 in blocking solution. Incubate for 1 hour with constant rocking.
  • Wash membrane three times with 1x TBST for 10 minutes each.

Signal detection

  • Prepare an ECL substrate according to the manufacturer’s instructions.
  • Incubate the membrane completely with the substrate for 1–5 minutes (adjust the time for more sensitive ECL substrates e.g. SuperSignal West Femto Chemiluminescent Substrate [Pierce]).
  • Expose the membrane to autoradiography film in a dark room or read using a chemiluminescence imaging system.

Line up the developed film in the correct orientation to the blot and mark the bands of the MW ladder directly onto the film. It is also advised to add notes such as lane content, film exposure time, and ECL properties.

Immunohistochemistry Technique

Immunohistochemical staining is a valuable tool for detecting specific antigens in tissues. In order to perform the standard staining procedure, first the tissue section has to be deparaffinized and then rehydrated before applying the primary antibody. Enzyme-conjugated secondary antibodies are then applied and the specific staining can be visualized after adding the enzyme-specific substrate. Occasionally, when weak or no staining is observed, an antigen “unmasking” by enzyme digestion, may be required.

The following procedure describes the application of peroxidase or alkaline phosphatase conjugates in the immunohistochemical labeling of formalin-fixed, paraffin-embedded tissue sections.

IHC Protocol Steps

  1. Slide Preparation
  2. Primary Antibody Reaction
  3. Secondary Antibody Reaction
  4. Substrate Preparation
  5. Development
  6. Counterstaining

Slide Preparation

Deparaffinization and Rehydration

  1. Place the slides in a 56-60 °C oven for 15 min. (Caution: Oven temperature must not exceed 60 °C).
  2. Transfer to a xylene bath and perform two changes of xylene for 5 min. each.
  3. Shake off excess liquid and rehydrate slides in two changes of fresh absolute ethanol for 3 min. each.
  4. Shake off excess liquid and place slides in fresh 90% ethanol for 3 min.
  5. Shake off excess liquid and place slides in fresh 80% ethanol for 3 min.
  6. Rinse the slides in gently running tap water for 30 seconds (avoid a direct jet which may wash off or loosen the section).
  7. Place in PBS wash bath for further rehydration (30 min. at room temperature)

Antigen Retrieval – Unmasking of Antigen

Note: This step is performed only in cases where weak or no staining occurs, or for antigens requiring “unmasking” according to the primary antibody specifications.

There are several possible ways for retrieval depending on the antibody and the antigen:

Enzyme retrieval:

  1. Apply 0.1% trypsin in PBS or 0.1% protease in PBS for 2-30 min. at 37 °C. Extending the incubation time may also enhance specific staining. Rinse in PBS for 10 min.

Microwave retrieval:

  1. Wash the slides with deionized H2O and place them in a microwave-resistant plastic staining jar containing antigen retrieval solution. Make sure slides are fully covered with solution.
  2. Operate the microwave oven for 5 min. on high power (~700 watts). Make sure slides are still covered with retrieval solution or add fresh solution and repeat microwaving.
  3. This process can be repeated 2-3 times.
  4. Let cool slowly at room temperature for at least 20 min. before proceeding to the next step.

Inactivation of Endogenous Peroxidase

Note: This step is performed only when using peroxidase-conjugated secondary antibodies or ExtrAvidin-Peroxidase.

  1. Place the slides on a flat level surface. Do not allow slides to touch each other. Do not allow the sections to dry out at any time.
  2. Add enough drops of 3% hydrogen peroxide to cover the whole section.
  3. Incubate 5 min. at room temperature.
  4. Rinse with PBS from a wash bottle.
  5. Place the slide in PBS wash bath for 2 min.

Primary Antibody Reaction

Note:

a. Pre-incubation of the sample with 5% BSA for 10 min. prior to the primary antibody reaction may decrease background staining. For best results with animal tissues, use 5 to 10% normal serum from the same species as the host of the secondary antibody.
b. Optimal dilution and incubation times should be determined for each primary antibody prior to use.

  1. Allow the slides to drain, shake off excess fluid with a brisk motion and carefully wipe each slide around the sections.
  2. Dilute the primary antibody or negative control reagent to its optimal dilution in diluent. The diluent alone may be used as a negative control. A positive control slide (a tissue known to contain the antigen under study) should also be run.
  3. Apply 100 µl primary antibody solution to the appropriate slides, covering the tissue sections.
  4. Tilt each slide in two different directions, so the liquid is spread evenly over the slide.
  5. Incubate for at least 60 min. at 37 °C in humidified chamber. Longer incubations are advised for low density antigens.
  6. Rinse gently with PBS from a wash bottle. Place the slide in a PBS wash bath for 5 min.

Secondary Antibody Reaction

Option 1 – Biotin/ExtrAvidin Detection

  1. Allow the slides to drain, shake off excess fluid and carefully wipe the slide as before.
  2. Dilute the biotinylated secondary antibody in diluent to its optimal concentration.
  3. Apply 100 µl to each slide, covering the tissue sections.
  4. Tilt each slide in two different directions.
  5. Incubate in a humidity chamber for at least 30 min. at room temperature.
  6. Rinse gently with PBS from a wash bottle.
  7. Place the slide in a PBS wash bath for 5 min.

ExtrAvidin Reaction

  1. Allow the slides to drain. Shake off excess fluid and carefully wipe the slide as before.
  2. Dilute ExtrAvidin peroxidase or ExtrAvidin alkaline phosphatase, in diluent to its optimal concentration.
  3. Apply 100 µl to all slides; cover the section.
  4. Tilt each slide in two different directions.
  5. Incubate in humidified chamber for at least 20 min. at room temperature.
  6. Rinse gently with PBS from a wash bottle.
  7. Place the slide in PBS wash bath for 5 min.

Option 2 – Enzyme-labeled Secondary Antibody

  1. Allow the slide to drain. Shake off excess fluid with a brisk motion and carefully wipe the slide as before.
  2. Dilute the peroxidase or alkaline phosphatase conjugated secondary antibody in the diluent to its optimal dilution.
  3. Apply 100 µl to all slides, covering the tissue sections.
  4. Tilt each slide in two different directions.
  5. Incubate 30 min. at room temperature or at 37 °C in humidified chamber.
  6. Rinse gently with PBS from a wash bottle.
  7. Place the slides in a PBS wash bath for 5 min.

Substrate Preparation

It is recommended to prepare the substrate mixture during the final wash step.

Development

  1. Allow each slides to drain. Shake off excess fluid and carefully wipe the slide as before.
  2. Apply enough drops of freshly prepared substrate mixture to cover the tissue section.
  3. Incubate 5-10 min. or until the desired color reaction is observed when monitored with the microscope. Terminate the reaction before background staining appears in the negative controls by rinsing gently with distilled water from a wash bottle.

Counterstaining

Note: When using AEC substrate, do not use alcohol-containing solutions for counter-staining (e.g., Harris’ hematoxylin, acid alcohol), since the AEC stain formed by this method is soluble in organic solvents. The slide must not be dehydrated, brought back to toluene (or xylene), or mounted in toluene-containing mountants.

  1. Apply enough Mayer’s hematoxylin to cover the section or place the slide in a bath of Mayer’s hematoxylin.
  2. Incubate for 0.5-5 min., depending on strength of the hematoxylin used.
  3. Rinse the slide gently with distilled water from a wash bottle.
  4. Rinse the slide under gently running tap water for 5 min. (avoid a direct jet which may wash off or loosen the section).
  5. Mount the sections using an aqueous mounting medium such as glycerol gelatin. Coverslip may be sealed with clear nail polish.

Flow Cytometry Protocol

This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different samples.

Preparation

  1. Harvest and wash the cells and determine the total cell number.
  2. Resuspend the cells to approximately 1×106 cells/mL in ice cold PBS. Add the appropriate volume of paraformaldehyde to obtain a final concentration of 4%.
  3. Fix for 10 minutes in a 37°C water bath.
  4. Wash cells twice with PBS containing 0.5% BSA.
    Note: For extracellular staining with antibodies that do not require permeabilization, proceed to step 8; for intracellular staining, proceed to step 5.

Permeabilization

  1. Add ice-cold 90% methanol (approximately 1mL per 1×106 cells) and vortex.
  2. Permeabilize for a minimum of 10 minutes on ice.

Immunostaining and Analysis

  1. Wash cells twice with PBS containing 0.5%BSA.
  2. Resuspend 1×106 cells in 100μL PBS containing 0.5%BSA.
  3. Add the primary antibody and incubate for 1 hour at room temperature (RT).
  4. Wash cells twice with PBS containing 0.5%BSA.
    Note: If using a fluorescent primary conjugated antibody, skip to step 14.
  5. Resuspend cells in fluorochrome conjugated secondary antibody diluted in PBS containing 0.5% BSA.
  6. Incubate for 30 minutes at RT.
  7. Wash cells twice with PBS containing 0.5% BSA.
  8. Resuspend cells in 0.5mL PBS and analyze on flow cytometer.

Direct ELISA Protocol

This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different samples.

Coating Antigen to Plate

  1. Coat the wells of a 96 well plate with 100μL of the desired antigen diluted in bicarbonate/carbonate solution. Include a serial dilution of the antigen for analysis.
  2. Cover plate with parafilm or plastic adhesive and incubate overnight at 4°C.
  3. Remove coating solution and wash plate 2 times with 200μL Phosphate-buffered saline +0.05% Tween20 (PBST). The solutions or washed are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel.

Blocking

  1. Block the remaining protein binding sites in the coated wells by adding 200μL of blocking buffer(1% milk/PBS or 1%BSA/PBS).
  2. Cover the plate and incubate for 2 hours at room temperature (RT).
  3. Wash plate 2 times with 200μL PBST.

Antibody Incubation

  1. Add 100μL of antibody diluted in blocking buffer.
  2. Cover the plate and incubate at RT for 2 hours.
  3. Wash plate 2 times with 200μL PBST.

Detection

  1. Add 100μL of the substrate solution per well.
  2. After sufficient color development add 100μL of stop solution to the wells.
  3. Read the absorbance of each well with a plate reader.

Analysis

  1. Prepare a standard curve from the data produced from the serial dilutions with concentration on the X-axis vs. absorbance on the Y-axis. Interpolate the concentration of the sample from this standard curve.

Indirect ELISA Protocol

Coating Antigen to Plate

  1. Coat the wells of a 96 well plate with 100μL of the desired antigen diluted in bicarbonate/carbonate solution. Include a serial dilution of the antigen for analysis.
  2. Cover plate with parafilm or plastic adhesive and incubate overnight at 4°C.
  3. Remove coating solution and wash plate 2 times with 200μL Phosphate-buffered saline +0.05% Tween20 (PBST). The solutions or washed are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel.

Blocking

  1. Block the remaining protein binding sites in the coated wells by adding 200μL of blocking buffer(1% milk/PBS or 1%BSA/PBS).
  2. Cover the plate and incubate for 2 hours at room temperature (RT).
  3. Wash plate 2 times with 200μL PBST.

Antibody Incubation

  1. Add 100uL of antibody diluted in blocking buffer.
  2. Cover the plate and incubate for 2 hours at RT.
  3. Wash plate 2 times with 200μL PBST.
  4. Add 100uL of conjugated secondary antibody diluted in blocking buffer.
  5. Cover the plate and incubate for 2 hours at room temperature.
  6. Wash plate 2 times with 200μL PBST.

Detection

  1. Add 100uL of the substrate solution per well.
  2. After sufficient color development add 100μL of stop solution to the wells.
  3. Read the absorbance of each well with a plate reader.

Anaylsis

  1. Prepare a standard curve from the data produced from the serial dilutions with concentration on the X-axis vs. absorbance on the Y-axis. Interpolate the concentration of the sample from this standard curve.

Sandwich ELISA Protocol

Coating with Capture Antibody

  1. Coat the wells of a 96-well plate with 100μL of the capture antibody diluted in bicarbonate/carbonate solution.
  2. Cover the plate with parafilm or plastic adhesive and incubate overnight at 4°C.
  3. Remove the coating solution and wash plate 2 times with 200μL Phosphate-buffered saline +0.05% Tween20 (PBST). The solutions and washes are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel or by aspiration.

Blocking

  1. Block the remaining protein-binding sites in the coated wells by adding 200μL blocking buffer per well.
  2. Cover the plate and incubate for 2 hours at room temperature (RT).
  3. Wash the plate 2 times with 200μL PBST.

Adding Samples

  1. Add 100uL of appropriately diluted samples and standards.
    Note: For accurate quantitative results, always compare signal of unknown samples against those of a standard curve. Standards (in duplicate and triplicate) and a blank must be run with each plate for analysis and to ensure accuracy.
  2. Cover the plate and incubate for 2 hours at room temperature.
  3. Wash the plate 2 times with 200μL PBST.

Incubation with Detection Antibody

  1. Add 100uL of diluted detection antibody to each well.
    Note: Be sure to check that the detection antibody recognizes a different epitope on the target protein to the capture antibody. This prevents interference with antibody binding.
  2. Cover the plate and incubate for 2 hours at room temperature.
  3. Wash the plate 4 times with PBS.
  4. Add 100μL of conjugated secondary antibody, diluted in blocking buffer immediately before use.
  5. Cover the plate and incubate for 2 hours at room temperature.
  6. Wash the plate 4 times with PBS.

Detection

  1. Add 100μL of the substrate solution per well.
  2. After sufficient color development add 100μL of stop solution to the wells.
  3. Read the absorbance of each well with a plate reader.
  4. Prepare a standard curve from the data produced from the serial dilutions with concentration on the X-axis vs. absorbance on the Y-axis. Interpolate the concentration of the sample from this standard curve.

Immunoprecipitation Protocol

This protocol is a recommendation only. Please optimize the procedure since experimental conditions can vary for different samples.

Sample Preparation

Non-Denaturing:

  1. Place cell culture dish on ice and wash the cells with ice cold Phosphate-buffered saline (PBS).
  2. Drain PBS and add ice cold lysis buffer.
  3. Scrape cells off the dish using a cell scraper and gently transfer the cell suspension into a microcentrifuge tube.
  4. Maintain agitation for 30 minutes at 4°C.
  5. Centrifuge in a micro centrifuge at 4°C. The time and force of centrifugation may vary depending on the cell type.
  6. Gently remove the tubes from the centrifuge and place them on ice.
  7. Aspirate the supernatant ad place in a fresh tube on ice.

Denaturing:

  1. Add 100uL denaturing lysis buffer to the cells.
  2. Mix well by vortexing vigorously for 2 to 3 seconds.
  3. Transfer the cell suspension to a microcentrifuge tube.
  4. Heat samples to 95°C for 5 minutes to denature.
  5. Dilute the suspension with 0.9mL of non-denaturing lysis buffer. Mix gently.
  6. Fragment the DNA by passing the lysed suspension 5-10 times through a needle attached to a syringe.
  7. Incubate on ice for 5 minutes.

Tissue lysates:

  1. Dissect the tissue quickly with clean tools. If possible, do so on ice to prevent degradation.
  2. Place tissue in microcentrifuge tubes and snap freeze by immersing in liquid nitrogen.
  3. Add 300uL of lysis buffer for approximately 5mg of tissue and homogenize with an electric homogenizer.
  4. Rinse twice with another 300uL of lysis buffer per rinse and maintain constant agitation for 2 hours at 4°C.
    Note: If denaturing is required, follow steps 2-5 from the denaturing protocol above.
  5. Centrifuge for 20 minutes at 12000rpm at 4°C in a microcentrifuge. Aspirate the supernatant and place in a fresh tube kept on ice.

Pre-clearing the Lysates

  1. Add 50uL of normal serum to 1 mL of lysate and incubate for 1 hour on ice.
  2. Add 100uL of bead slurry to the lysate and incubate for 30 minutes at 4°C.
  3. Spin in microcentrifuge at 14,000 x g for 10 minutes at 4°C.
  4. Discard bead pellet and keep supernatant for immunoprecipitation.

Immunoprecipitation with Antibody in Solution

  1. While on ice, add 10-50ug cell lysates to a microcentrifuge tube plus the recommended amount of antibody.
  2. Incubate the sample with the antibody for a few hours at 4°C with gentle agitation.
    Note: The length of incubation depends on the amount of protein and the affinity properties of the antibody.
  3. Prepare the Sepharose beads.
  4. Mix the slurry well and add 70-100uL of the beads to each of the samples, while on ice.
  5. Incubate the beads mixture for 4 hours at 4°C with gentle agitation.
  6. Centrifuge tube after incubation and wash 3 times with lysis buffer.
  7. Add 25-50 uL 2x loading buffer and boil for 5 minutes at 100°C.
  8. Centrifuge and transfer the supernatant to a new tube for Western blotting.

FAQs

Can you provide the exact immunogen sequence for an antibody?

The immunogen sequence is not readily available for many of our antibodies. We invest a great deal of time and expertise in designing our antibodies to deliver the most effective products.

If you require the immunogen sequence for your experiments, please contact our Scientific Support team at technical@stratech.co.uk

How are antibodies produced?

Polyclonal production

Polyclonal antibodies are made by immunizing with an antigen. Repeated immunizations of the same antigen at intervals of several weeks stimulates specific B cells to produce large amounts of the anti-antigen. The blood will contain a variety of antibodies, each to a different epitope on the antigen. The immune-sera can be used in its crude form, where high levels of specific antibodies are present, or the specific antibodies can be isolated from sera components by affinity purification.

Monoclonal production

To produce monoclonals the same immunization protocol is used and all antibody-forming cells (e.g. B cells) are removed. These are fused with immortal tumor cells to become hybridomas, which are screened for antibody production and performance.

The hybridomas that produce antibodies are given clone names, which are uniquely assigned to permit identification. The antibody producing hybridoma cells are cloned by isolation and cultivated using tissue culture. Alternatively, genes coding for antibody production can be cloned into transfection vectors to produce recombinant antibodies.

Unlike polyclonal antibodies, monoclonal are homogenous with defined specificity to one epitope. The antibody secreted by the cells into the culture media can be harvested and used in its crude form, or it can be purified by affinity chromatography.

How do you purify the antibodies?

Huabio Antibodies can be supplied in several forms, ranging in purity.

Various methods are used to purify our antibodies include:

  • Protein A/G Purification
  • Protein Affinity Purification
  • Peptide Affinity Purification

Review the datasheet for more information.

What are recombinant antibodies?

Recombinant antibodies are produced in vitro by cloning antibody genes for immune-specific heavy and light antibody chains into high-yield expression vectors. These vectors are then introduced into expression hosts (eg bacteria, yeast, or mammalian) to generate the recombinant monoclonal antibodies. Recombinant antibodies can be used wherever you would normally use a traditional monoclonal antibody.

Recombinant antibodies offer several advantages over both traditional monoclonal and polyclonal antibodies:

  • Improved consistency and reproducibility
    Because recombinant antibodies are developed from a unique set of genes, antibody production is controlled and reliable. Several problems with hybridoma production can be avoided, such as gene loss, gene mutations, and cell-line drift. This leads to antibodies with very little batch-to-batch variability, giving you highly reproducible results.
  • Improved sensitivity and specificity
    With recombinant technology, it is easier to improve both antibody specificity and sensitivity through antibody engineering. The selection process for the desired clone occurs at both the hybridoma and recombinant cloning stages, allowing us to select the most favorable antibody qualities.
  • Ease of scalability
    With the antibody genes isolated, antibody expression can be carried out at any scale and in a shorter timeframe than traditional monoclonal technology. This means we can generate tailored antibodies in weeks rather than months.
  • Animal-free high-throughput production
    Once the antibody-producing gene is isolated, animal-free in vitro production can be implemented. For antibodies generated using our phage display technology, even the gene of the antibody can be isolated with an animal-free procedure.

Optimizing antibody dilutions

The optimal antibody concentration, or optimal working dilution, is the one that gives the best staining with the minimum background; this must be determined experimentally for each assay.

Many of our antibodies have dilution factors included on the datasheet under the ‘Applications with Dilution’ section. However, these can require some optimization, as we describe in this article. Varying the relative concentrations of an antibody and an antigen solution can control the extent of antibody-antigen complex formation. As it is not usually possible to change the concentration of the antigen, the optimal antibody concentration must be determined for each application and set of experimental conditions.

How do I optimize the amount of antibody to use from a given dilution factor?

The optimal concentration is usually determined using a series of dilutions in a titration experiment.

A titration experiment is done by first selecting a fixed incubation time, and then a series of experimental dilutions for the antibody. If a product datasheet suggests using a 1:200 dilution, it is recommended to test dilutions of 1:50, 1:100, 1:200, 1:400 and 1:500. Test each of these chosen concentrations on the same sample type to determine which one works best.

Many antibodies will have a similar batch-to-batch consistency, so in most cases, only one titration experiment is required. However, if there is a change in the results of the staining between batches of the same antibody, we recommend performing another titration experiment. Such variation may be more common with polyclonal antibodies.

Choose a positive control

If you require a suitable positive control, please check the datasheet, which will often have a suggested positive control. Always ensure the tissue or cell line you use is from a tested species. Not all the datasheets will have a suggested suitable control, and we recommend the following in these circumstances:

  • Check to see the validation images of the antibody. Any tissues, cells or lysates that have been used successfully by these experiments can be considered a suitable positive control.
  • Try looking at the Swiss-Prot or Omnigene database links on the datasheet. These databases will often have a list of tissues that the protein is expressed in. These can also be considered suitable positive controls.
  • Check the GeneCards entry for the protein. This will usually provide you with relative levels of expression in various tissues.
  • If you still have difficulty finding a suitable control, we recommend doing a quick literature search on PubMed to see which tissues and cells express the protein of interest.

What are the shipping conditions?

We select the most appropriate shipping condition to ensure the product’s stability during transit.

  • Blue ice (also called wet ice) — products will arrive with blue ice packs to keep them cool (approximately 4ºC). If the ice pack has thawed by the time the product reaches you, this should be perfectly fine and will not affect product quality.
  • Ambient temperature — some of our products are stable enough to be shipped at room temperature, so they will not come with any dry ice or ice packs. We only ship products at room temperature if we have experimentally determined that this will not affect product quality during shipping.

How should I store my antibody?

For many of our antibodies, you should aliquot and freeze at -20°C or -80°C. Aliquoting minimizes damage due to freezing and thawing and reduces possible contamination.

Centrifuge the vial at 10,000 x g for 20 seconds—aliquot at least 10 μL into low-protein-binding microcentrifuge tubes. Antibody aliquots should be frozen and thawed only once, with any remainder kept at 4°C.

It is essential to follow the recommendations on the datasheet.

How do I avoid freeze/thaw damage?

We suggest storing our antibodies at -20°C unless otherwise stated on the datasheet.

Repeated freeze/thaw cycles can form protein aggregates, denaturing an antibody, reducing its binding ability. Consider pipetting the antibody into smaller aliquots and thawing as needed. Antibody aliquots should be frozen and thawed only once, with any remainder kept at 4°C.

Should I still use the product if the ice pack is warm?

We ship products at different temperatures depending on how sensitive they are to heat.

If the ice pack is thawed and the temperature inside the box rises to greater than 4°C, please be assured that this should not affect the product’s stability. The antibodies are sufficient to use if you store them correctly upon receipt.

We ship all products at temperatures they are known to be stable at, but it’s critical to store them as directed by the datasheet upon arrival. The best conditions for long-term product stability can differ from the optimal short-term conditions we use during shipping.


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