DETECTION METHODS FOR WESTERN BLOT

Western Blot is a technique commonly used in scientific research laboratories. By means of this technique, the different proteins present in a sample are separated according to their molecular weight by gel electrophoresis, and are subsequently transferred to a membrane to proceed with their identification using specific antibodies.

Although the foundation of the technique remains, over the years new detection methods have been developed in order to obtain more accurate results and also allow a quantitative analysis of proteins.

In this post we bring you a summary of the main detection methods for Western Blot with the advantages and disadvantages of each of them.

DETECTION METHODS FOR WESTERN BLOT

1.- RADIOACTIVE DETECTION METHODS
This type of detection was one of the first methods used to reveal the results of the Western Blot, by labeling the antibodies with radioactive conjugates.

The main advantage of this method lies in its sensitivity, but it has the great drawback that when using radioactive materials there is a risk to the researcher’s health and safety. Furthermore, it is a high-cost technique and its execution is time consuming.

Radioactive detection is not currently among the Western Blot detection methods of choice. In fact, its use is discouraged.

2.- ENZYMATIC DETECTION METHODS
These methods are based on the use of secondary antibodies conjugated to an enzyme that catalyzes a reaction with a specific substrate.

Within this category, detection can be carried out by means of two types of enzymatic reactions:

2.1 COLORIMETRIC DETECTION
In this case, the enzyme bound to the secondary antibody triggers a reaction with the substrate giving rise to a colored precipitate that can be visually identified.

The advantages of this method lie in its speed, simplicity and low economic cost, in addition to not requiring any special equipment. Its drawback is its low sensitivity (in the order of picograms).

This method is usually used when it is necessary to quickly and easily analyze the presence or absence of a certain protein.

2.2 DETECTION BY CHEMIOLUMINISCENCE
In chemiluminescence assays, the enzyme bound to the secondary antibody triggers a reaction with a luminescent substrate generating light.

In this case, the great advantage is the high sensitivity provided by this method (in the order of femtograms), allowing proteins with very low levels of expression to be identified. As a drawback, note that it requires the use of specialized equipment to read the results.

3.- FLUORESCENT DETECTION METHODS
This type of detection is based on the use of secondary antibodies conjugated to fluorophores that produce signal by themselves, without the need to add any additional substrate.

Among the advantages, it should be noted that the signal is more stable than that produced by enzymatic detection methods and, above all, that the possibility of using fluorophorized antibodies with different emission wavelengths on the same Western Blot membrane allows multiplexing the experiments. It should also be noted that this method also allows quantifying the protein present in the sample.

As disadvantages, a lower sensitivity than chemiluminescence detection, and the need to use specialized equipment.

Fluorescence is among the most widely used Western Blot detection methods today.

ANTIBODIES FOR IMMUNOFLUORESCENCE

The immunofluorescence (IF) technique, based on the detection of a specific antigen of interest by using fluorescently labeled antibodies, is a technique widely used in research laboratories due to its simplicity and reliability.

The results can be visualized by fluorescence microscopy using short wavelengths and, in addition to detecting the presence or absence of a certain protein in the sample, it is possible to determine its distribution in the sample or confirm the presence of post-translational modifications, among others.

In this post we bring you some keys related to antibodies for immunofluorescence that can help you optimize the results of your tests.

3 KEYS WHEN USING ANTIBODIES FOR IMMUNOFLUORESCENCE

1.- THE IMPORTANCE OF THE SPECIFICITY OF ANTIBODIES FOR IMMUNOFLUORESCENCE
As in any other immunoassay, the specificity of the primary antibody against our target antigen is a determining factor in the reliability and success of the results. The more specific the antibody, the better the signal obtained and the less background noise generated.

Let us also remember that an antibody that has a high specificity against an antigen in a certain technique does not have to do so in another, even if it is the same antigen. Hence the importance of validating each antibody for each technique in which it will be used. In the case at hand, it is essential to previously validate the immunofluorescence antibodies to be used in the assay.

How can we validate the antibodies for immunofluorescence? For there are various methods such as positive and negative expression experiments using, for example, knock-out cell lines, by experimental manipulation of the location of the target protein, protocol optimizations, etc. Or, resorting to commercial antibodies already validated for use in this technique.

2.- CONTROLS FOR IMMUNOFLUORESCENCE
The inclusion of controls, as in any other experiment, will increase confidence in the results obtained in terms of specificity and sensitivity.

To avoid errors derived from autofluorescence phenomena or from nonspecific binding of antibodies, the use of negative controls in immunofluorescence assays is very important.

Additionally, additional controls such as omission of the primary antibody, the use of isotype controls and of negative and positive cell lines for the antigen of interest may be included.

3.- DILUTION OF THE ANTIBODIES FOR IMMUNOFLUORESCENCE
To optimize the results of the tests, another key point is the titration of the antibodies to determine the ideal dilution to use in each case. This will also vary depending on whether we are dealing with a purified antibody or an antiserum.

In this sense, it is important to achieve a good signal / background noise ratio, that is, an optimal relationship between the intensity of the fluorescent signal from the antigen of interest and the background signal due to nonspecific junctions. If we apply the primary antibody at a very low concentration, it will be very difficult to distinguish the positive signal. Conversely, an overly concentrated antibody will excessively increase background noise.

The typical concentration / dilution ranges for immunofluorescence experiments are usually between 1-10ug / mL in the case of using purified antibodies, and between 1: 100 – 1: 1000 for the antisera.

In this post you can remember some other recommended dilutions for other techniques and immunoassays.

Stress and the autoimmune diseases

What stress could to to our body
Stress

Psychological factors Happen to Be Thought to play a leading role in the disposition, beginning, or class of various physical disorders. Stress is defined as a disorder that occurs when someone perceives the requirements of a scenario that surpasses his tools and can raise the body’s vulnerability to specific ailments, Implementing an immunosuppressive effect. An upgrade consists of the consequences of emotional strain in these diseases which are directly correlated with immunological mechanisms like infections, autoimmune disorders and neoplasms, in addition to its impact on cardiovascular ailments. It’s noted that timely emotional interventions might help regulate the stress response and enhance health behavior, teaching people more adaptive procedures to translate life’s challenges with much more powerful answers.

Stress and autoimmune diseases

The Majority of the Signs that stress Results in The beginning and course of autoimmune disorder are circumstantial as well as also the mechanics by which stressful events influence autoimmunity aren’t entirely understood. But, there are studies which have demonstrated a link between stress and autoimmune disorder.

Many autoimmune disorders share 2 common features: Immune System dysregulation and anxiety pathways. two pathways, the HPA axis and the sympathetic nervous system (SNS) regulate the immune response through the release of corticosteroids and norepinephrine (NE), respectively. These neuroimmunomediators behave on immune cells like macrophages through the alpha or beta adrenergic receptors on the face, to regulate the creation of significant regulatory cytokines, and normally act to inhibit inflammation. But under certain conditions, NE promotes inflammation during conversation with macrophage alpha-1 adrenergic receptors and the subsequent increase in production of tumor necrosis factor alpha (TNF-a). Even though macrophages do not ordinarily express the receptors, their saying on the plasma membrane of macrophages and monocytes happens in certain disease conditions. Through these mechanisms, the HPA axis as well as the SNS affect the course and development of rheumatoid arthritis (RA), which likely play significant roles in its pathogenesis. Thus, therapeutic agents acting on the modulation of neural pathways which normally govern Immune System homeostasis, can be good for the treatment of RA and other autoimmune disorders. Additionally, it has been demonstrated that a disorder of the neuroendocrine system might be among those risk factors associated with the pathogenesis of rheumatic ailments. Persistent inflammatory pressure mediated by neural and humoral signals during the active condition of this disease and autoantibodies from the arrangements of the neuroendocrine system might also take part in neuroendocrine dysfunction.

Effect of stress on autoimmune disease

Stress and it's consequences
Stress and it’s consequences

The best proof of this impact of pressure on autoimmune thyroid disorder is that the association between the onset of Graves’ hyperthyroidism and improved anxiety. But, there aren’t many reports of a potential connection between tension and Hashimoto’s thyroiditis, likely because the beginning and development of the disorder are normally insidious and also the consequences of anxiety could go undetected. The pathogenesis of Graves’ ophthalmopathy is unknown, but the presence of an inflammatory response from the orbital tissues, linked to the activity of antithyroid antibodies, was shown. Lately it’s been clarified that ophthalmopathic facets might be determined by environmental variables, one of which pressure stands outside, which supports the hypothesis that the autoimmune procedures within this bronchial inflammation may be associated with environmental aspects. Patients experiencing hypothyroidism can occasionally experience anxiety attack, intense stress, palpitations, and eventually become quite emotionally obese people generating increased anxiety. This disorder, in which the thyroid gland is hypoactive, is often accompanied by exhaustion, fainting, and various levels of melancholy.

In research studies, in which numerous ecological elements such as stressful life events were researched, it was reasoned that emotional strain, measured as psychosocial stress from the household, seems to be involved in the induction or progression of diabetes-related autoimmunity from the youth, because of a union of hormonal levels and neurological signals that affect insulin sensitivity and desire, in addition to Immune system.

Viral Vectors

Types of Viral Vectors
VIRAL VECTORS

Viruses have proven to be one of the most efficient vehicles in transferring genetic information to eukaryotic cells. This is precisely why molecular biologists have put so much effort into modifying their genome to make them more secure (incompetent for replication, attenuation) but maintaining their ability to transfer and express recombinant genetic material. Thus, viral vectors have become a very useful tool in biomedical research both in vitro (cell cultures) and in vivo (animal experiments, gene therapy) but we must be aware that these improvements in intrinsic biosecurity of viral vectors and their commercial availability can facilitate a relaxation in the application of safe practices, circumstance that must be avoided.

Groups of viral vectors

Viruses from which the viral vectors are derived may be assigned to danger group 1 (eg adeno-associated virus, baculovirus), group 2 (eg adenovirus, herpesvirus, poxvirus) but there are also representatives to group 3 (e.g. HIV). Generally, the level of biosecurity required to work with the viral vectors is that of NCB2 but this may vary according to the proposed experimental procedure (e.g. large-scale production, animal inoculation) or the biological activity of the transgene (eg oncogene, biotoxin). ).

To correctly evaluate the risk in working with viral vectors, the following must be considered:

  • the danger group of the unmodified parental virus;
  • the degree of modification made to obtain the defective vector;
  • the function of inactivated viral genes;
  • host range and pseudotyping;
  • its ability and efficiency to integrate into the genome of the host cell
  • the function of the transgene.

The lab offers all research staff working with viral vectors a specific face-to-face course and informative material that can be of great help in conducting a correct risk assessment in each case. Among this material we can highlight the pathogen technical sheets (FTP), available on the intranet, and the following fact sheets:

  •     Viral vectors and biosecurity level
  •     Viral vector biosecurity level and cellular functions
  •     Biosecurity and viral vectors
  •     Biosecurity and lentiviral vectors
  •     Acting in case of accidental exposure to lentiviral vector