General

Western Blot FAQ

The Western Blot is an immunoassay for the detection of proteins in complex samples that is carried out following 4 sequential steps:

  • SDS-PAGE (polyacrylamide gel electrophoresis) to separate proteins.
  • Protein transfer to a nitrocellulose or polyvinylpyrrolidone membrane.
  • Incubation of the membrane with a specific antibody against the protein of interest.
  • Detection of antigen-antibody binding.

In this post we bring you a compilation of frequently asked questions about Western Blot that can help you answer questions and improve the performance of your immunoassay.

1.- How many times can I use the antibody solutions once they are prepared?

It is recommended that you always use freshly formulated antibody solutions. In the event that previously made solutions are reused, it is of utmost importance to ensure that no bacterial overgrowth has occurred, especially in cases where the solution has previously been blocked with a blocking agent.

2.- Why do intense bands appear at higher or lower molecular weights than expected?

On some occasions, even using the antibodies at the lowest recommended dilution, it binds to proteins whose bands come out well below or well above the actual molecular weight of the protein of interest. In most of these cases, these bands correspond to isoforms of the protein in question, or to the formation of dimers.

A search is recommended to see if any isoforms are described in the literature or if the protein dimerizes. The use of a different antibody can also be tested.

3.- If you use more than one primary antibody, in what order should they be incubated?

Both simultaneous incubation with all primary antibodies and successive incubation with each is possible.

4.- Is it necessary to measure the protein concentration in the sample before doing the Western Blot?

It is not an essential step, although it is recommended to adjust the amount of sample to be loaded in the gel. The determination of the total protein concentration can be carried out by the BCA method .

5.- How can I separate and transfer proteins with sizes greater than 200kDa?

You can find several tips for transferring large proteins in this post .

6.- How can I avoid background noise?

This is one of the most common western blot frequently asked questions. Background noise may be due to causes as disparate as too high an antibody concentration, nonspecific binding of the secondary antibody, cross-reactions of the antibodies with the blocking agent or insufficient washes, among others.

Here we tell you how to solve these problems in Western Blot.

7.- Is Western Blot a quantitative immunoassay?

The Western Blot is not a quantitative method, since a standard curve for the protein of interest is not usually performed in each blot.

8.- Why are the Western Blot bands different in size than expected?

Although the separation of proteins in the Western Blot is based on their size, there are other variables that can influence the migration speed through the gel, and cause the observed band to differ from what could be predicted based on the actual size of the protein.

The most influencing factors are:

  • Post-translational modifications
  • Post-translational splits
  • Isoforms and other variants
  • Relative load

9.- How much sample should I load in the gel?

The amount will depend on the type of sample we handle:

  • Cell lysates : the amount should be optimized based on the expression levels of the protein of interest in each case, but in general we can load between 20 and 30 ug of total protein per well.
  • Purified protein : 10 to 100 ng of protein are usually loaded.

10.- Why can’t I detect my recombinant protein?

It could be because the recombinant protein that is expressed in the sample does not include the antigenic sequence recognized by the antibody that we are using, or in the case that the recombinant protein is expressed with a tag and it is very bulky or interferes with the antigenic sequence, could prevent its binding to the antibody.

In the case of working with recombinant proteins, it is always recommended to include an endogenous positive control in the Western Blot.

11.- Should I use milk or BSA as a blocking agent?

In general, BSA will give cleaner results since by containing fewer proteins, the probability of cross-reactions with the antibody is reduced.

However, in certain cases, blocking with milk will work better precisely because a greater variety of blocking proteins has the ability to block a greater range of different proteins.

12.- Why do so many bands appear in the Western Blot?

This may be due to several factors, including:

  • The antibody is not specific enough for the target protein.
  • Antigen degradation by proteolysis.
  • Too much protein per lane.
  • Overly sensitive detection system.
  • Ineffective blocking.
  • Antigen concentration too low.

13.- What is the difference between a Western Blot in reducing and non-reducing conditions?

To perform a Western Blot under reducing conditions, a reducing agent such as DTT or B-mercaptoethanol is added to the sample buffer to break the disulfide bridges, whereby the protein will be in its denatured form when the immunoassay is performed. .

14.- Should I use reducing or non-reducing conditions in my test?

Western Blots are usually performed under denaturing conditions. In any case, it is advisable to consult the technical sheet of the antibodies to ensure that they will work against the denatured protein.

General

Glossary Of Antibodies

Research work in the laboratory with antibodies and immunoassays involves familiarizing yourself with a number of specific terms around immunology, immunoglobulins, and the immune response.

In this entry we bring you a short glossary of antibodies that includes the basic and fundamental terminology to better understand the functioning and applications of immunoglobulins in the research laboratory.

ADJUVANT

Chemical compound that is added to the antigen to increase its immunogenicity and thus stimulate the animal’s immune response for the production of antibodies. Freud’s complete / incomplete adjuvants and aluminum hydroxide are among the most common adjuvants.

AFFINITY

Measure of interaction or binding between the antigen and the antibody.

ANTIBODIES

Proteins produced by B lymphocytes of the immune system, also known as immunoglobulins, that identify, bind, and help destroy antigens in a highly specific way.

CONJUGATE ANTIBODY

Antibodies chemically linked to fluorochromes or chromogens to enable visual detection of them.

CAPTURE ANTIBODY

Antibody with which the ELISA plate is covered and which binds to the antigen contained in the sample to be applied later.

DETECTION ANTIBODY

Primary antibody that is used in the sandwich ELISA and that specifically binds to the immobilized antigen.

HUMANIZED ANTIBODY

Genetically engineered antibody where a minimal part of a murine antibody (5-10%) is introduced into a human antibody (90-95%) in order to minimize the response of the human immune system against them.

MONOCLONAL ANTIBODY

Homogeneous population of antibodies produced by a single B lymphocyte clone that specifically recognize a single epitope of the antigen.

MURINE ANTIBODY

Mouse antibody.

POLYCLONAL ANTIBODY

Heterogeneous solution of antibodies produced by different B lymphocytes that recognize different epitopes of the same antigen.

PRIMARY ANTIBODY

Antibody that binds directly to the antigen of interest.

CHIMERIC ANTIBODY

Genetically engineered antibody by fusing parts of a murine antibody (33%) with parts of a human antibody (67%).

SECONDARY ANTIBODY

Conjugated antibody that binds to the primary antibody that recognizes the antigen of interest.

THERAPEUTIC ANTIBODY

A single clone of a specific antibody produced by a cell line that is administered for therapeutic purposes. Therapeutic antibodies can be murine, chimeric, humanized, or fully human.

ANTIGEN

Substance that arouses a specific immune response.

ANTISERUM

Serum from an immunized animal containing the antibodies of interest.

APTAMER

Affinity reagents with antibody-like applications, which specifically bind to the antigen of interest. Unlike antibodies, aptamers are produced in vitro and can be made up of peptides or nucleic acids.

AVIDITY

Measurement of the binding strength of the antigen-antibody complexes.

LIGHT CHAIN

A polypeptide subunit of an immunoglobulin located in each of the arms of the Y-shaped structure (each antibody contains two identical light chains). It has two subdomains: the constant region and the variable region that intervenes in binding to the antigen.

HEAVY CHAIN

Polypeptide subunit of an antibody that defines its isotype. It consists of a constant region (which will vary depending on the immunoglobulin class) and a variable region that is involved in binding to the antigen.

CARRIER

Large, highly antigenic molecule that is conjugated to small antigens to induce a more effective and specific immune response in producing antibodies.

AFFINITY CONSTANT

Numerical value indicating the binding strength between the antigen and the antibody.

OPTIMAL WORKING DILUTION

Antibody concentration with which the maximum positive signal and the minimum background noise and nonspecific signal are achieved.

EPITOPE

The specific region of the antigen that is recognized and to which the antibody binds.

SPECIFICITY

Ability of an antibody to bind only to the desired antigenic determinant.

FAB SHARD

The Fab or antigen-binding fragment is each of the 2 arms of the Y-shaped structure of the antibody. It is obtained after enzymatic digestion of the antibody with papain.

HAPTENE

Small molecules that are only capable of arousing an immune response if they are linked to a Carrier protein.

HYBRIDOMA

Cell line resulting from the fusion of antibody-producing B lymphocytes with an immortalized tumor line (myeloma).

HOST (HOST SPECIES)

Animal species in which the antibodies are generated.

IMMUNOGENICITY

Ability of an antigen to induce the production of antibodies.

IMMUNOGEN

Substance capable of inducing an immune response.

IMMUNOGLOBULINS

Protein families that function as antibodies.

ISOTYPES

Immunoglobulin classes depending on the heavy chains they have.

ANTIBODY LIBRARY

Method to obtain antibodies by creating an ex vivo repertoire of immunoglobulins that can be screened against a specific antigen.

ASCITES FLUID

Fluid extracted from the abdomen of the host animal that contains monoclonal antibodies produced by the hybridomas previously inoculated in the animal.

MYELOMA

Bone marrow tumor that can be adapted to grow indefinitely in cell culture.

PRE-ADSORPTION

Adsorption of the antiserum with proteins or serum of different species to eliminate the antibodies that can give rise to cross reactions.

AFFINITY PURIFICATION

Purification of the antibody against the specific antigen it recognizes.

PURIFICATION BY PROTEIN A / G

Class-specific purification to isolate all Immunoglobulins of a certain isotype, regardless of their affinity for the antigen of interest.

REACTIVITY

Species from which the epitope used in immunization was derived, or those with high homology for that sequence.

CROSS REACTIVITY

Binding of the antibody to similar epitopes of other proteins or antigens.

VARIABLE REGION

Region containing the antigen binding site.

PRE-IMMUNE SERUM

Serum withdrawn before immunization that is used as a control.

TITLE

Assay to determine the optimal concentration of an antibody for a specific application.

General

Immunogenetics of the NKG2D ligand gene family.

NKG2D ligands (NKG2DLs) are a group of major histocompatibility complex (MHC) class I-like molecules, the expression of which is induced by cellular stresses such as infection, tumorigenesis, heat shock, tissue damage, and DNA damage. They act as a molecular danger signal alerting the immune system for infected or neoplastic cells. Mammals have two families of NKG2DL genes: the MHC-encoded MIC gene family and the ULBP gene family encoded outside the MHC region in most mammals.

Immunogenetics of the NKG2D ligand gene family.
Immunogenetics of the NKG2D ligand gene family.

Rodents such as mice and rats lack the MIC family of ligands. Interestingly, some mammals have NKG2DL-like molecules named MILL that are phylogenetically related to MIC, but do not function as NKG2DLs. In this paper, we review our current knowledge of the MIC, ULBP, and MILL gene families in representative mammalian species and discuss the origin and evolution of the NKG2DL gene family.

Natural killer (NK) cells are key effectors in cancer immunosurveillance and can be used as a prognostic biomarker in diverse cancers. Nonetheless, the role of NK cells in pancreatic cancer (PC) remains elusive, given conflicting data on their association with disease prognosis.

In this study, using conventional K562 target cells and complementary engineered target cells providing defined and synergistic stimulation for NK cell activation, a correlation between impaired NK cell cytotoxic degranulation and PC progression was determined. Peripheral blood mononuclear cells (PBMCs) from 31 patients with newly diagnosed PC, 24 patients with non-malignant tumors, and 37 healthy controls were analyzed by flow cytometry.

The frequency, phenotype, and effector functions of the NK cells were evaluated, and correlations between NK cell functions and disease stage and prognosis were analyzed. The results demonstrated that effector functions, but not frequency, of NK cells was progressively decreased on a per-cell basis during PC progression. Impaired cytotoxic degranulation, but not IFN-γ production, was associated with clinical features indicating disease progression, such as high serum CA19-9 and high-grade tumors. Significantly, this impairment correlated with cancer recurrence and mortality in a prospective analysis.

Furthermore, the impaired cytotoxic degranulation was unrelated to NKG2D downregulation but was associated with increased circulating and tumor-associated TGF-β1 expression. Thus, NK cell cytotoxic activity was associated with PC progression and may be a favorable biomarker with predictive and prognostic value in PC.

Bystander cells enhance NK cytotoxic efficiency by reducing search time

Natural killer (NK) cells play a central role during innate immune responses by eliminating pathogen-infected or tumorigenic cells. In the microenvironment, NK cells encounter not only target cells but also other cell types including non-target bystander cells. The impact of bystander cells on NK killing efficiency is, however, still elusive. In this study we show that the presence of bystander cells, such as P815, monocytes or HUVEC, enhances NK killing efficiency.

With bystander cells present, the velocity and persistence of NK cells were increased, whereas the degranulation of lytic granules remained unchanged. Bystander cell-derived H2O2 was found to mediate the acceleration of NK cell migration. Using mathematical diffusion models, we confirm that local acceleration of NK cells in the vicinity of bystander cells reduces their search time to locate target cells.

In addition, we found that integrin β chains (β1, β2 and β7) on NK cells are required for bystander-enhanced NK migration persistence. In conclusion, we show that acceleration of NK cell migration in the vicinity of H2O2-producing bystander cells reduces target cell search time and enhances NK killing efficiency.

HNSCC subverts PBMCs to secrete soluble products that promote tumor cell proliferation

The immune system detects shifts from homeostasis and eliminates altered cells. However, neoplastic cells can modulate the host response to escape immunosurveillance thereby allowing tumor progression. Head and neck squamous cell carcinoma (HNSCC) is one of the most immunosuppressive cancers but its role in co-opting the immune system to actively promote tumor growth has not been investigated.

In this study, we investigated the influence of soluble factors secreted by HNSCC and non-neoplastic epithelial cells on proliferation, apoptosis, activation, cytokine gene expression and phenotypic polarization of immune cells of healthy donors. Then, we determined if the immunomodulation caused by HNSCC-derived soluble products leads to immunosubversion by assessing proliferation, migration and survival of tumor cells exposed to soluble products secreted by modulated immune cells or co-cultured with immune cells.

Soluble products from HNSCC inhibited proliferation and cytokine expression in PBMCs, activation of T cells, and polarization of CD4+ towards the Th17 phenotype. These changes co-opted the immune cells to favor cell proliferation, survival and migration of HNSCC.

This immunosubversion was observed both indirectly with secreted products and with direct cell-to-cell contact. We conclude that HNSCC-derived secreted products create an immunosuppressive environment that facilitates evasion of tumor cells and subverts the immune cells into a pro-tumoral phenotype.

NK and NKT cells in the diagnosis of diffuse lung diseases presenting with a lymphocytic alveolitis

Diffuse lung diseases (DLD) are characterized by different immunophenotypes in the bronchoalveolar lavage fluid (BALF). We aimed to evaluate the diagnostic value of BALF NK and NKT cell counts of patients with DLD and lymphocytic alveolitis.We assessed 202 patients with DLD, who underwent BALF immunophenotyping. Samples were routinely processed by flow cytometry and lymphocyte subsets were compared between patients with sarcoidosis (n = 106), hypersensitivity pneumonitis (HP; n = 53), and other DLDs (n = 43).

We compared absolute counts and percentages of NK and NKT cells between patients with HP versus the remaining DLD patients. To assess the accuracy of BALF lymphocyte subsets in the diagnosis of HP, we calculated the respective areas under the receiver operating characteristic curves (AUC-ROC).RESULTSPatients with HP had significantly higher numbers of BALF NK cells, and its percentage was significantly associated with a higher odds of HP, even after adjustment for the NKT and CD8+ cells.

For the absolute number of BALF NK cells, we found an AUC-ROC of 0.76 (95%CI = 0.68-0.84) when comparing patients with HP versus the remaining DLD. The cut-offs of 2000 NK cells/mL and of 2.4% NK cells in the BALF had a specificity and a negative predictive value over 80% for the diagnosis of HP. BALF NK cells absolute counts were significantly higher in HP patients with a restrictive pattern.

No such differences were observed for NKT cells.BALF NK immunophenotyping may be a helpful adjunct to the diagnostic work-up of DLD, particularly in the differential diagnosis of HP.

Functions of natural killer cells, General, Human mesenchymal stem cells modulate allogeneic immune cell responses

Functions of natural killer cells

Natural killer (NK) cells are effector lymphocytes of the innate immune system that management a number of varieties of tumors and microbial infections by limiting their unfold and subsequent tissue injury.

Recent analysis highlights the truth that NK cells are additionally regulatory cells engaged in reciprocal interactions with dendritic cells, macrophages, T cells and endothelial cells. NK cells can thus restrict or exacerbate immune responses. Although NK cells would possibly seem like redundant in a number of situations of immune problem in people, NK cell manipulation appears to carry promise in efforts to enhance hematopoietic and stable organ transplantation, promote antitumor immunotherapy and management inflammatory and autoimmune problems.

Functions of natural killer cells
Functions of natural killer cells

Inflammatory monocytes and macrophages have been recognized as key gamers within the pathogenesis of atherosclerosis, arterial hypertension, and myocardial infarction (MI). They develop into highly effective mediators of vascular irritation via their capability to secrete and induce the manufacturing of proinflammatory cytokines, chemokines and adhesion molecules and thru the manufacturing of reactive oxygen species primarily by way of their NADPH oxidase.

Interplay of NK cells and monocytes in vascular irritation and myocardial infarction.

Importantly, a crosstalk exists between NK cells and monocytes that works by way of a feedforwad amplification loop of T-bet/Interferon-gamma/interleukin-12 signaling, that causes mutual activation of each NK cells and monocytes and that fosters recruitment of inflammatory cells to websites of irritation. Recently, we have now found that this crosstalk is essential for the unrestricted improvement of angiotensin II (ATII) induced vascular harm in arterial hypertension, an important danger issue for atherosclerosis and heart problems worldwide.

In this evaluate, we may even talk about potential implications of this interaction between NK cells and monocytes for the pathogenesis of coronary atherosclerosis and myocardial infarction and potential therapeutic choices.

Natural killer (NK) cells are lymphocytes of the innate immune system that secrete cytokines upon activation and mediate the killing of tumor cells and virus-infected cells, particularly people who escape the adaptive T cell response brought on by the down regulation of MHC-I.

The induction of cytotoxicity requires that NK cells contact goal cells via adhesion receptors, and provoke activation signaling resulting in elevated adhesion and accumulation of F-actin on the NK cell cytotoxic synapse. Concurrently, lytic granules endure minus-end directed motion and accumulate on the microtubule-organizing middle via the interplay with microtubule motor proteins, adopted by polarization of the deadly cargo towards the goal cell.

Ultimately, myosin-dependent motion of the lytic granules towards the NK cell plasma membrane via F-actin channels, together with soluble N-ethylmaleimide-sensitive issue attachment protein receptor-dependent fusion, promotes the discharge of the lytic granule contents into the cleft between the NK cell and goal cell leading to goal cell killing.

Herein, we are going to talk about a number of disease-causing mutations in major immunodeficiency syndromes and the way they affect NK cell-mediated killing by disrupting distinct steps of this tightly regulated course of.

Human immunodeficiency syndromes affecting human natural killer cell cytolytic exercise

Many organic processes depend on the power of cells to measure native ligand focus. However, such measurements are constrained by noise arising from diffusion and the stochastic nature of receptor-ligand interactions.

It is thus essential to know how precisely, in precept, focus measurements will be made. Previous theoretical work has principally investigated this in 3D beneath the simplifying assumption of an unbounded area of diffusion, however many organic issues contain 2D focus measurement in bounded domains, for which diffusion behaves fairly in a different way.

Here we current a principle of the precision of chemosensation that covers bounded domains of any dimensionality. We discover that the standard of chemosensation in decrease dimensions is managed by area measurement, suggesting a common precept relevant to many organic programs. Applying the speculation to organic issues in 2D exhibits that diffusion-limited signalling is an environment friendly mechanism on time scales according to behaviour.

Background. A Kampo drugs, Shahakusan (SHS), has been prescribed in late part of an infection that causes inflammations within the lung. But impact of SHS on viral an infection in respiratory tract has by no means been reported. Objectives. To consider anti-influenza virus exercise of SHS and its mode of actions via immune programs. Methods.

SHS (0.three g/kg/day) was orally administered to BALB/c miceforupper (URI) or decrease respiratory tract an infection (LRI) of influenza virus A/PR/8/34. The virus titer of nasal lavage fluid (NLF) at 5 or 2 day postinfection (p.i.) and cytokine mRNA expressions in mandibular lymph node or lung at 5 or Four day p.i. had been evaluated for URI or LRI, respectively.

The histopathological examinations of lung tissue and NK cell exercise within the splenocytes had been additionally evaluated at Four day p.i. on LRI. Results. When SHS was administered from 7 days earlier than to Four days p.i. for URI, the virus titer was considerably decreased compared with water-treated management, and IL-4, IL-1β, and IL-10 mRNA expression was decreased, however IL-12A mRNA expression was elevated. Administration of SHS from in the future earlier than to in the future p.i. for LRI considerably decreased the virus titer.

SHS additionally decreased infiltration of inflammatory cells within the bronchoalveolar areas and injury of desquamated mucosal epithelia of bronchiole, decreased IP-10 mRNA expression, and elevated NK cell exercise. Conclusion. SHS has no direct impact on influenza virus an infection however exerts antiviral impact in mice by its immunomodulating exercise via motion of NK cells and anti inflammatory exercise within the lung.

HIV controllers: a multifactorial phenotype of spontaneous viral suppression

A small minority of HIV-infected people, generally known as HIV controllers, is ready to exert long-term management over HIV replication within the absence of therapy. Increasing proof means that the adaptive immune system performs a essential function on this management but additionally {that a} mixture of a number of host and/or viral elements, fairly than a single trigger, results in this uncommon phenotype. Here, we evaluate current advances within the research of these outstanding people.

We summarize the epidemiology and scientific traits of HIV controllers, and subsequently describe contributing roles of host genetic elements, innate and adaptive immune responses, and viral elements to this phenotype. We emphasize distinctive traits of HIV-specific CD4 T cell responses and of CD4 T cell subpopulations which might be steadily present in HIV controllers. We talk about main controversies within the subject and the relevance of the research of HIV controllers for the event of novel therapeutic methods and vaccines.