Antibodies - Science topic

Dear Dr. YINGJI JIN I am glad that you have understood.

You're welcome.

Best Wishes.

Maybe check the blocking buffer? always use fresh made one or frozen stocks. Or does the Super-ECL reagent is fresh made? Or check the expose machine, do you use the correct filter?

Try Wormbook (http://www.wormbook.org/chapters/www_introreversegenetics/introreversegenetics.html#d0e928).

If you are asking about using an anti-GST antibody to immunopurify or detect a GST-tagged protein from cells, my guess is that the epitopes of the monoclonal antibodies have been selected so that they recognize the GST tag and not the endogenous GST. Since the epitope of each monoclonal antibody consist of a small number of amino acids, it should not be too difficult to develop such a selective antibody.

Take the sequences of an antibody containing the framework combination you wish to use, e.g. human Vkappa1/human VH3 consensus for a well-folding and stable framework,

(DIQMTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRT, EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARWGGDGFYAMDYWGQGTLVTVSS).

Replace the original CDR sequences in this antibody sequences with your own CDR sequences (see https://plueckthun.bioc.uzh.ch/antibody/Numbering/SeqHeader.html , https://plueckthun.bioc.uzh.ch/antibody/Numbering/Numbering.html for a comparison of different CDR definitions to match the one corresponding to the sequences you have).

You can then submit the resulting sequences to an antibody modelling server, e.g. https://opig.stats.ox.ac.uk/webapps/sabdab-sabpred/sabpred/ to generate a 3D model

Agree with Yannick. However, some antibodies work for both WB and imaging applications. A relatively easy way to test a primary ab for imaging is usually by light (fluorescence) microscopy, using applicable secondary ab (fluorophor conjugated). Also keep in mind that most ab do not bind after fixation with standard glutaraldehyde concentrations used for EM.

Dear Ana,

As Dr Sudeep stated, the blockinf step os importante tô reduce unspesific ligations. However, more importante in some cases os the use of isotype controls/the secondary antibody that could adress unspesific internacional with the fluorophore. Especially, tandem fluorophores can be metabolized by some cells (DOI: 10.1002/cyto.a.20774.)

Dear Dr. Malgorzata Krok

In Western Blot when you decide to choose the primary antibody make sure that the primary is specific to the protein of interest and should be of a different host species than the sample. Also, the primary antibody should be validated for use in western blotting, the information for which will be made available in the package insert for the antibody which you will purchase.

Another point to remember is that the primary should be specific for either the denatured or native conformation of the protein of interest, which will depend on the type of PAGE (either SDS or native) you perform.

Further, choose a secondary antibody directed against the species of the primary antibody. So, you will need a secondary antibody that is raised in a species different than the host species of the primary antibody. For instance, if your primary antibody is raised in a mouse, you will need an anti-mouse secondary antibody raised in goat, rabbit, etc.

If you would want to perform multiplex Western Blot, use primary antibodies from different host species for each target being probed. Ideally, use a combination of antibodies from two distantly related species such as rat and rabbit, avoiding the combinations like mouse and rat or goat and sheep. This will help in the selection of appropriate secondary antibodies to minimize antibody cross reactivity that could affect your end results.

Regards,

Malcolm Nobre

For complex samples such as cell extracts, the sandwich ELISA may be more sensitive than the indirect ELISA because it captures all of the antigen onto the surface, no matter how little of the antigen is present, until the capture antibody is saturated, and everything else is washed away.

For the indirect ELISA, the sensitivity is limited by the proportion of the sample that consists of the antigen of interest. There is a limited binding capacity of the surface, and everything in the sample competes for the limited surface.

So, for the indirect ELISA, you probably need to use higher antigen concentrations for the standard curve than you would for the sandwich ELISA.

Camila Román

Western Blotting can be influenced by a variety of factors, and it can sometimes be challenging to pinpoint the exact issue. Here are some key areas to consider:

Antibody Species and Host Origin:

Numerous causes might be to blame for this kind of problem. What I can recommend is that you lengthen your blocking period to at least 2 hours and that you avoid incubating the secondary antibody for longer than 2 hours.

Use freshly prepared buffers and make sure about the proper transfer. Hope you will get a good result next time.

Saroj Kumar Khan Samir Ranjan Panda

Hi Saroj and Samir,

I have been experiencing very variable levels of cross reactivity with the highly cross preadsorbed mouse thermo antibodies so I was looking for a better alternative.

Do you have a working positive control?

Are you sure you're seeing growing bacteria, not precipitate of some kind? TBS alone can hardly support bacterial growth, and you even have it supplemented with NaN3. By the way, people rather use 0.01M or 0.1% NaN3 instead of 0.01%. If you are concerned, you may increase NaN3 concentration.

10.1136/gutjnl-2016-312141

Please follow pub med article

This idea is theoretically feasible. Live-cell imaging requires a high-quality fluorescence microscope and precise experimental procedures. The general process (incubation, washing, imaging) is similar to routine steps. To achieve better results, fine-tuning and optimization of the details are necessary through experimentation.

Thank you both! Labkit thus far seems to be working the best for a trainable segmentation.

Have a look at this database:

Maybe you can find those clones.

I agree with Malcolm Nobre, perhaps this table will help you with future conversions.

Good morning,

I'm having the same problem, did you get any idea on how could we enhace cytokine's production please ?

Thank you

Hello Wang Xiang

The EVs have surface proteins present on their surface via which the IgG will bind. This interaction is an antigen-antibody interaction. All antigen-antibody bonds are weak physical bonds. The forces involved are not strong covalent bonds but rather they are weaker bonds such as electrostatic interactions, hydrogen bonds, and the Van der Waals forces.

I came across the article attached below which may interest you.

As per the paper, in pancreatic ductal adenocarcinoma, IgG in circulation binds to the tumor neoantigens, melanoma associated antigen B1 (MAGE B1) on circulating EVs released by tumor cells.

Best.

Yes, we work with hundreds of cell lines, and multiple antibody. First we expand the cell line. Then clarify and concentrate with a TFF system to simplify the process for Purification.

The best way for this is to use the MALDI-TOF MS configuration. For ESI top-down or native-MS-like approach is needed for the analysis of either heavy or light chains. You may combine SEC and native analysis for ESI-LCMS or gas phase fragmentation of antibody light chain to select SRM-like produced peptide for quantification. ESI produces multiple charge states for large molecules therefore occurring charge envelopes reduce the precursor intensity if the top-down quantification is aimed. MALDI gives reduced charge states thus either the identification or quantification approach would be more easy/more practical if the sample is not a protein complex but a purified antibody.

Rather than the selection of MS or acquisition technique, it is more important to choose the sample prep strategy herein. Garbage in garbage out for any MS system. What is your consideration about the sample prep and what is your sample matrix? how would you purify/clean, reduce, and fractionate your sample? It is more critical to assess prior to MS detection. Otherwise many interfering compounds, and protein peptides make your quantification worse and that is why it is recommended to use MRM and signature proteolytic peptide identification is more appropriate to perform absolute quantification...

Did you remove albumin? Pass diluted serum through an albumin depletion resin and observe the supernatant...Preparing a highly diluted sample and ultracentrifugation to overcome viscosity following the application to the immunoaffinity column for custom-made antibody purification may also work without an albumin removal strategy as mentioned earlier ...

Thanks

The answer in this article :

Hi KK

CD3/CD28 antibodies activate T cells in a more directed manner. OVA peptide active OT-1 cells must be presented by APC.

There are two aspects to the response to this question:

1 - requirements for immunogenicity, necessary to get a good immune response in the context of a natural immune system

2 - requirements for the specific molecular interactions between antigen and antibody to achieve sufficiently high affinity and specificity

Normally, to get a good immune response, your antigen needs to comprise both B-cell epitopes and T-cell epitopes. The antibodies produced by the B-cell, through differential splicing, also contribute the recognition unit of the B-cell receptor, needed for B-cell activation. B-cell receptor mediated uptake of the antigen. A protein antigen will be proteolytically processed and its peptides displayed by MHC to TH2 T-helper cells. Stimulatory signals from the T-helper cells will trigger somatic hypermutation (affinity maturation), antibody class switch, B-cell proliferation and differentiation into antibody-producing plasma cells and long-lived memory B-cells. Highly polyvalent antigens and antigens that act as B-cell mitogens via Toll-like receptors can activate B-cells in a T-cell independent manner.

By covalently coupling non-proteinacious antigens (Haptens) to a carrier proteins, an immunogenicity is created that combines a polyvalent display of the Hapten with the T-cell epitopes of the carrier protein, allowing these conjugates to induce a robust immune response.

2 - requirements for high affinity and specificity

The interaction energy between antigen and antibody and thereby the binding affinity is limited by the contact surface. Small Haptens are rarely bound with high affinity, and high affinity interactions between antibodies and Haptens are characterized by a binding mode in which the Hapten is deeply buried in the VL/VH interface, thereby maximising the contact surface. While hydrophobic interactions, aka the reduction of solvent exposed hydrophobic surface area, provide the driving force for the binding interaction, they are fairly unspecific. For good specificity, hydrogen bonding and charge interactions are needed.

Wolfgang Schechinger Finally, i got the 150 linearity with single point calibration. Thanks for your reply.

Are you using a new blocking agent that might contain free biotin that would compete with the antibodies?

Hello Smriti Chand

1. Yes, you are right. B-actin is also present in the nucleus, as a component of chromatin remodeling complexes. You may use α-tubulin as a marker for the cytoplasmic fraction.

The soluble α/β-tubulin is considered to be a reservoir of subunits that are available for microtubule polymerization in the cytoplasm, but there are reports supporting the presence of tubulin in the nucleus of cultured animal cells. The reason for nuclear accumulation of tubulin, which does not occur under normal circumstances, is a process of pathophysiological significance and may represent a defense mechanism against stress or malignant transformation. This is the reason why nuclear tubulin may be detected only in cancer or in transformed cells.

2. Calnexin is highly abundant in ER as well as the outer nuclear membrane. It has also been found at ER-mitochondria contact sites. But the possibility of contamination of nuclear fraction with membrane fraction cannot be ruled out. Nevertheless, you may try caveolin-1 for the membrane fraction. Caveolin-1 is a plasma membrane protein that has a fundamental role in the formation of caveolae and acts as a connective protein that organizes the macromolecular complexes on the cell surface which participates in intracellular signaling.

3. In addition to fibrillarin, you could use histone (H3) for the nuclear fraction. H3, one of the core histones along with H2A, H2B and H4 is known to form nucleosomes with nuclear DNA. It plays a crucial role in activating the spindle assembly checkpoint in response to a defect in mitosis.

You may try these markers.

Hope it helps!

Best.

Hi Mickael Pereira, I usually use CD64'CD68'CD32 and CD11b to in both cases and agree with Louis-Chales Beland

Sir, virus neutralization test can be performed.

Dear Dr. Denny Kollareth

The protocol that you have shared could have potential negative effects on data quality. The process of stripping and re-probing can take a considerable amount of time and reagents. Moreover, the membrane stripping procedure may remove sample proteins from the membrane. Therefore, quantification of phosphoproteins following membrane stripping would not be ideal.

I would suggest that the above drawbacks from the traditional chemiluminescent method of detection could be overcome by using fluorescent western blotting which will allow you to detect both versions of the protein (multiplexing) on the same membrane.

Multiplexing your western blot typically require antibodies against the total and phosphorylated protein raised in two different species. Fluorescent secondary antibodies bearing two different fluorophores (with non-overlapping spectra) can be used allowing for multiplexing which means both phosphorylated and non-phosphorylated species can be detected simultaneously.

Another important aspect is to select antibodies which are highly specific for the phosphorylated protein state only. If the antibody is not specific, this can result in simultaneous detection of total protein and confound the interpretation of your results.

Other factors that you should consider while detecting phosphoproteins are:

1. The phosphoprotein must be kept intact. For this reason, protease and phosphatase inhibitor cocktails must be added to cell lysis buffer prior to use. These will help prevent proteins from being degraded and dephosphorylated. Keep the lysate always on ice.

2. You should avoid using milk for blocking the membrane because milk contains casein, an abundant phosphoprotein that can cause high background. Use BSA instead.

3. You should avoid using phosphate-based buffers like PBS as it can interact with anti-phospho antibodies, affecting the binding of the antibody to the target protein. Use Tris-based buffers, for instance, Tris-buffered saline with Tween-20 (TBST) instead.

4. Use proper controls.

Regards,

Malcolm Nobre

It's possible that the cells could have undergone changes in their machinery, which could affect translation, folding, or post-translational modifications of the antibodies. Cell lines can exhibit genetic instability or undergo phenotypic changes over time, especially when cultured for extended periods or subjected to passaging. These changes could potentially impact protein expression and functionality.

Using a relatively low passage number from the initial commercial stock (P.7) should minimize the risk of significant changes, but it doesn't entirely rule out the possibility. Other factors, such as variations in cell culture conditions, media components, or reagents, could also contribute to the observed issues.

To troubleshoot this problem, you may consider these:

1. Verify the integrity of your cell line by comparing it to a freshly obtained sample from the same commercial source or obtaining a new batch altogether.

2. Assess the stability of your cell culture conditions, including media, supplements, and any other factors that could impact cellular behavior.

3. Explore alternative antibody production methods or cell lines to confirm whether the observed changes are specific to the current system.

After incubation with primary antibody overnight try soaking the membranes in TBST for 30 mins followed by washing while rocking for one hour. this will decrease the back ground Sonia Marina

The bright spots are something in your samples and not a blotting artifact. My best guess is that it is just an accumulation of small peptide fragments from proteolysis of the proteins in your lysate. They give a strong signal because they may include peptides recognized by your primary antibody.

You might try adding protease inhibitors very early in the preparation of your samples and see if that helps.

1. please try to increase your fixation time to 10-12 mins.

2. Use permeabilization step with 0.25%Triton x100 for 10 min.

3. Block with 2-3% BSA made in PBS.

4. Use a dilution of 1:200 for your antibody.

I think this will solve your problem.

Best wishes

Subhajit

Sourcing animal tissue samples, especially those genetically modified to express fluorescent proteins such as GFP (Green Fluorescent Protein), RFP (Red Fluorescent Protein), and tdTomato can be somewhat challenging due to ethical considerations, storage conditions, and potential legal restrictions. Here are some potential sources:

Louis-Charles Béland Thank you for your advice, I will try.

Hi Berkay Alpay, it is not uncommon to observe variations in the molecular weight of protein bands detected by different antibodies, even when targeting the same protein. These variations can arise due to various factors such as antibody specificity, post-translational modifications, or isoform-specific differences. In the case of GAT-1 (SLC6A1), different antibodies may recognize different isoforms or post-translationally modified forms of the protein, resulting in variations in the detected molecular weight. Additionally, differences in sample preparation and experimental conditions can also influence the observed band migration.

To address this issue and reconcile the differences in molecular weight, you can consider the following steps:

Yes, antibodies for immunofluorescence of frozen sections and cells can be shared. Antibodies used in immunofluorescence experiments might be shared among researchers or colleagues in order to conduct comparable experiments or validate results. Sharing antibodies enables replication and validation of findings, fostering scientific transparency and collaboration in the research community.

Hi Bonnie,

We got you! Send us the peptide or protein sequences and we will make suggestions for your antigen candidate. Once the project is set, we will synthesize the peptide and then use it for immunization.

Our deliverable will include the purified antibody AND the peptide that we use in the antibody production!

Send us a request with your project details to initiate our collaboration: https://www.biomatik.com/services/custom-antibody-service/polyclonal-antibody-production.html

We look forward to hearing from you soon.

Best,

Biomatik Team

homo Arginine Elisa kit.

Wolfgang Schechinger There are. But, I wanted to interact with someone who has done it in real time. Published articles sometimes have too much information, yet not the minute details.

Dear Laetitia,

for DOTA, PEG (or poly amino-acid) spacered TFP-esters might be the most straightforward approach. Maleimides are tricky here, as you need to partially reduce the antibodies to get free sulfhydryls.

Same for NOTA, but ready to use linker/ligands might be difficult to procure, as it might be the case for DFO. PubChem and SciFinder might be helpful to locate them, at least to retrieve their CAS-Numbers.

Georgiy Leonov that's only partially correct, while the excitation spectrum of PI is so broad that it overlaps with everything, the emission spectra are very well separated. Using proper filters would allow to detect the two stains independently.

see also my answer to Huang's new question: https://www.researchgate.net/post/Is_it_possible_to_do_the_double_staining_of_anti-beta_tubulin_antibody_and_propidium_iodidePI_staining_for_the_co-culture_of_PC12_and_C6_cells3

Activation of naive T cells generally requires antigens to be presented by dendritic cells in lymph nodes. Unlike the cells of the innate immune system, T cells and B cells can identify specific features of pathogens. Therefore, B or T cells can be raised against pathogens not detected by the innate immune system.

Tumor necrosis factor TNF alpha1

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LT2-TNFR2 cross also TNF TNFR2 and And LT2- TNFR1

Cypzox is best medicine 💊 for these arthritis various.

The red clouds e.g. are even present, if we strip the blot completely and scan again or don´t even incubate the membrane with antibodies at all....

Hi Shubhita,

check the guide from University of South Florida:

I'm keeping my fingers crossed for your success of rabbit immunization.

Dear @Romain Gioia, this could happen if the first polyAbs are against the peptide from the whole target protein. In WB an endogenous protein may mask this epitope/peptide. The other reasons are already mentioned.

Please don't hesitate to watch

Sheetal Bhardwaj

you can search here for your antibody:

I'll surely try with dry blot technique. Thank you James

The ability of large molecule antibodies, produced in response to synthetic GMO mRNA infiltration, to cross the blood-air barrier and protect the lungs from Covid-19 is uncertain. While antibodies can be effective in neutralizing viral particles, the blood-air barrier is a specialized interface designed to protect the delicate lung tissue. The passage of large molecules, such as antibodies, through this barrier is generally limited. However, advancements in drug delivery systems and nanotechnology may offer potential strategies to enhance the delivery of therapeutic agents across this barrier. It is important to note that the specific characteristics and mechanisms of synthetic GMO mRNA and the resulting antibody response would need to be thoroughly studied and validated to determine their ability to cross the blood-air barrier and provide protection against Covid-19 in the lungs.

The duration for which IgM antibodies last after vaccination against COVID-19 can vary from person to person. IgM antibodies are typically the first type of antibodies produced by the immune system in response to an infection or vaccination. However, their presence tends to be temporary, usually persisting for a few weeks to a couple of months.

For COVID-19 vaccination, most vaccines primarily induce the production of IgG antibodies, which provide longer-lasting immunity compared to IgM antibodies. IgM antibodies, if present after COVID-19 vaccination, are often transient and gradually wane over time.

It's important to note that the specific duration of IgM antibody presence after COVID-19 vaccination may depend on factors such as the individual's immune response, the vaccine type, and the specific characteristics of the vaccine formulation. Ongoing research is being conducted to better understand the duration and dynamics of antibody responses following COVID-19 vaccination.

It's worth mentioning that while IgM antibodies may decline, the immune system's memory B cells and T cells continue to play a crucial role in maintaining long-term immunity against COVID-19. These cells can recognize the virus and mount a rapid immune response upon re-exposure, even in the absence of detectable levels of IgM antibodies.

To determine the duration of immunity and the need for booster shots, researchers and health authorities are closely monitoring vaccinated individuals and conducting studies to evaluate antibody responses and immune memory over extended periods of time.

If you have specific concerns about your antibody response after COVID-19 vaccination, it is recommended to consult with healthcare professionals or refer to guidance from reputable health organizations.

Hello Colin, I found an antibody for you.

This antibody is useful for mouse and human tissue. If you are working with other species either you try it and found ount whether it works on your tissue or you try to find another antibody with the help of the biocompare-platform

Good luck!

Hm. What about cryptic splice sites? Not sure how to detect that, maybe trying to rescue the sequence by PCR and sequence the shorter bands, if any.

Or some unexpected signal sequences.

But all that is just wild guessing.

The Biotin-Streptavidin interaction is one of the strongest non-convalent interactions known to science. I would not expect that free biotin is able to dissociate the complexes. That's why desthio-biotin and other reagents have been developed, where the interaction with streptavidin is weaker and dissociation indeed may be achieved by adding biotin.

The potential problem that I can see is free biotin (e.g. bound to albumin) in the culture medium, that will happily and irreversibly cover all the binding sites of the streptavidin and outcompete the antibodies. Biotin-free BSA is available to make up serum free media, but quite expensive.

That's why I'd react the antibodies first with the streptavidin beads, but it's easy (and reasonable) to investigate and possibly to demonstrate that the order really matters.

If you use NHS-Biotin (or similar) to label the antibodies, you might get low binding to strep, as this handle is frequently too short for the biotin to reach the binding pocket. Biotin-C6-NHS (AKA Biotin-X-Osu) with an aminohexanoic acid spacer facilitates the binding to streptavidin. For more sophisticated and longer spacers (hydrophilic poly-ethyleneglycoles, poly-sarcosins, etc., you may find useful information in this brochure by Iris Biotech: https://www.iris-biotech.de/global/biotinylation-reagents

Cytoplasmic expression of antibody fragments in E. coli often results in the production of aggregates within inclusion bodies. Antigen binding activity can be reconstituted after polypeptide refolding1,2 but recovery takes a hit in this process

Hello, I would recommend an NKX2.5 antibody from CUSABIO: https://www.cusabio.com/Polyclonal-Antibody/NKX2-5-Antibody-1033304.html The quality is approved by many researchers.

Heating with excess tris can reverse aldehyde modifications of amino groups (imine formation), so I'd just try and set up a series of experiments e.g. perfoming PCRs or whatever you want to do with the DNA later.

Altenatives to PFA could be methanol or ethanol.

In addition to what was mentioned, I think the most probable cause is the high voltage you used, try to start with 50 volt for 25 min this would allow the bands to stack together and then proceed with 150 volt.

Dextran-conjugated fluorescent dyes (e.g. Texas red or fluorescein) are also possible markers. I don’t know that dextrans have no effects on physiology, but doing control injections with only marker and no antibody should be informative, whichever marker you eventually settle on.

No, this is the wrong technique in this situation. Desalting is only going to exchange the buffer, and you will still have the exact same mixture of antibody and carrier protein. Most of your A280 signal is probably due to carrier protein If you want to isolate the antibody, you are going to have to do protein A purification or possibly ion exchange or hydrophobic interaction chromatography to remove the carrier. The pure Ab will still need to be exchanged into PBS by desalting or dialysis.

It is a 14kDa protein, but runs a little higher on Western blots because it doesn't bind to SDS in an exact 1:1 ratio. On my blots, it is usually at around 18kDa.

First, blank the spectrophotometer. You do this with a solution that has ALL of the components that you would expect to find in your sample EXCEPT the molecule of interest. So, if you want to measure antibody that is in PBS/EDTA your blank has to be PBS/EDTA.

However you should also consider how the sample has been prepared. If you desalted the antibody into the PBS/EDTA buffer, the blank is the column equilibration buffer. If you dialysed the Ab into buffer, the strictly correct blank to use is the spent dialysis buffer (not fresh buffer). If you diluted 10mg/ml Ab in 50 mM Tris/0.1 % azide into PBS/EDTA to give a 1mg/ml Ab solution, the strictly correct blank to use then is a 1/10 dilution of the tris/azide buffer in the PBS/EDTA buffer, so that the ONLY difference between the blank and sample is the presence of the molecule that you wish to measure.

Finally, you will also need to use a cuvette that can transmit UV wavelengths e.g., quartz.

In addition to the above responses, you may also test glycine alone and non-denaturing detergents such as triton x-100 at low concentrations to disrupt the oligomerization affinity. But you should be aware that, some of the abovementioned suggestions (including triton) will hamper affinity-induced protein purification...Therefore upon breaking the aggregate, techniques such as SEC, UF, Dialysis, gravity flow desalting/buffer exchange may get rid of these agents prior to successful affinity chromatography.

As you have 20% FBS in your culture, there should be tons of bovine IgG in your prep, and you thus might enrich bovine IgG.

Can you resort to immobilized anti-mouse instead? Or even immobilized antigen, if available?

please read whole the answer .

Yes, you can use a hot-start polymerase for amplifying PCR products before sequencing. Hot-start polymerases are designed to minimize non-specific amplification and primer-dimer formation during PCR setup by blocking the polymerase activity at lower temperatures. This feature helps to improve the specificity and efficiency of PCR amplification.

Using a hot-start polymerase can be particularly beneficial when amplifying PCR products for sequencing, as it helps to reduce background noise and improve the quality of the sequencing results. By preventing non-specific amplification, the hot-start polymerase can help ensure that the amplified product corresponds to the intended target sequence, leading to more accurate sequencing data.

There are different types of hot-start approaches available, including antibody-based methods and chemically modified polymerases. Antibody-based hot-start polymerases typically use antibodies to block the polymerase activity at lower temperatures, which is then released during the initial denaturation step of PCR when the temperature is raised. This ensures that the polymerase becomes active only at the optimal temperature for PCR amplification.

It's important to note that the choice of polymerase depends on various factors, including the specific requirements of your experiment and the compatibility of the polymerase with the sequencing method you plan to use. Therefore, it is recommended to carefully select a hot-start polymerase that is compatible with your specific sequencing protocol and follow the manufacturer's instructions for optimal performance.

Using a hot-start polymerase that is bound to an antibody for nested PCR amplification should not interfere with the subsequent sequencing analysis. The antibody that blocks the polymerase activity at lower temperatures is designed to be released and activate the polymerase at the optimal temperature for PCR amplification.

Once the PCR amplification is complete and the desired fragment of the viral genome is amplified, the antibody-bound polymerase will have been fully activated and catalyzed the synthesis of the PCR product. At this point, the polymerase will no longer be bound to the antibody and any remaining antibody will not interfere with the subsequent steps, including sequencing.

For Sanger sequencing, the amplified PCR product is typically purified to remove any remaining primers, dNTPs, enzymes, and other contaminants. The purification process, such as using spin columns or enzymatic purification kits, will effectively remove the antibody and any other residual components of the PCR reaction. The purified PCR product can then be used as the template for Sanger sequencing, and the sequencing reaction will proceed without interference from the hot-start polymerase or its antibody.

However, it's always a good practice to confirm the compatibility of your specific hot-start polymerase and antibody combination with the downstream sequencing method you plan to use. You may want to consult the manufacturer's guidelines or perform some pilot experiments to ensure that the hot-start polymerase you have and its associated antibody do not interfere with the sequencing analysis.