Month: July 2020

Tips To Validate The Antibodies

The antibodies are one of the most widely used tools in research laboratories to identify, isolate or block various molecules. Due to the specificity assumed, it is essential to validate the antibodies for use in each of the techniques in which they are going to be applied, since, as we have seen on other occasions, an antibody can be very specific, for example in an ELISA immunoassay , and yet give a very weak signal in other types of tests such as Western Blot .

Validating the antibodies implies testing the following aspects:

  • Antibody specificity in the selected technique
  • Affinity for antigen
  • Reproducibility between trials

In this post we collect some tips to guide you when validating the antibodies and defining their specificity.

Main Problems With Antibodies, How To Solve Them?

The most common problems that arise from the use of antibodies are cross-reactivity, variability between batches and their application in an incorrect technique.

1.- CROSS REACTIVITY

  • Problem: Antibodies not only bind to the target antigen, but also to other proteins present in the sample.

2.- VARIABILITY BETWEEN LOTS

  • Problem: It is more frequent in the case of polyclonal antibodies, since the same antibody produced in different animals could work slightly differently.

3.- WRONG APPLICATION

  • Problem: The different techniques and experimental conditions in which each one is carried out can modify the folding of the target protein, and consequently, its ability to bind to the antibody.

To avoid any of these problems, it is vitally important to validate the antibodies before use.

But how do you do it? Pay attention to the trials we list below.

Assays To Validate The Antibodies

Antibodies can be validated by a multitude of functional tests, each of which has its advantages and disadvantages:

ELISA

  • Pros:
    • Quantitative test, allows confirmation of sensitivity.
    • High performance assay, a high number of samples can be processed simultaneously.
  • Cons:
    • It does not determine whether the antibody can lead to cross reactivity.

WESTERN BLOT (WB)

  • Pros:
    • Qualitative test, easy and simple to carry out.
    • Ideal for denatured proteins.
  • Cons:
    • Difficult to optimize.
    • It takes a lot of time.
    • Only a few antibodies can be tested at the same time.

IMMUNOHISTOCHEMISTRY (IHC) / IMMUNOCYTOCHEMISTRY (ICC)

  • Pros:
    • Relatively low-cost techniques.
    • Qualitative test.
  • Cons:
    • Difficult to standardize.
    • The accessibility of epitopes can vary in fixed tissues.
    • It does not determine whether the antibody non-specifically recognizes other proteins with the same cell location.

FLOW CYTOMETRY (FC)

  • Pros:
    • High performance assay, a high number of samples can be processed simultaneously.
    • Easy to optimize.
  • Cons:
    • It does not determine whether the antibody can lead to cross reactivity.

KNOCKDOWN siRNA

  • Pros:
    • KnockDown cell lines can be used in all assays: WB, IHC / ICC, FC …
  • Cons:
    • KnockDown is transitory.
    • Difficult to optimize.

KNOCKOUT CELL LINES (KO)

  • Pros:
    • They guarantee that there will be no expression of the target gene, so they are excellent negative controls, guaranteeing the specificity of the antibody.
    • They can be used in all tests: WB, IHC / ICC, FC …
  • Cons:
    • KnockOut cell lines against specific genes are not always viable.

MASS SPECTROMETRY (MS)

  • Pros:
    • High performance assays, a high number of samples can be processed simultaneously.
  • Cons:
    • Requires the use of a mass spectrometer and qualified personnel.
    • Difficult to optimize.

Other Considerations To Take Into Account To Validate The Antibodies

1.- The choice and preparation of cell lines or tissue samples , using the corresponding positive and negative controls, and taking into account whether the antibody recognizes the protein in its native or denatured form.

2.- The protocols , paying special attention and optimizing the incubation times individually (very short periods can affect sensitivity, and on the contrary, excessive incubation times could result in background noise problems), dilutions of work, blocking conditions, etc.

3.- The choice of buffers , determining the optimum in each case.

Ionic Channels And Cancer Immunology

The ion channels are membrane proteins that control the passage of ions therethrough in response to different stimuli. The dysregulation and / or dysfunction of these ion channels is related to a multitude of diseases, including cancer.

Cancer development depends on the effectiveness of the immune system in destroying tumor cells, and the key steps in this process are regulated by various ion channels .

These channels can affect the activation of immune cells and their toxicity against cancer, making them a more than interesting target for research and development of new antitumor therapies.

The Role Of Ion Channels In Cancer Immunology

Tumor cells are recognized by the immune system through the specific antigens they express on their surface, and through joint and integrated action between innate and adaptive immunity mechanisms, these cancer cells are killed. But some of them are capable of developing mechanisms to reduce their immunogenicity, and therefore, not to be recognized by the immune system and thus avoid their eradication.

Strategies to enhance anti-cancer immunity could be directed towards increasing efficiency in antigen presentation, recognition and elimination of tumor cells by the immune system, or inhibition of tumor-induced immunosuppression. Ionic channels play a fundamental role in any of these processes, as the following examples show:

  • Lysis of tumor cells by CD8 + cytotoxic T cells is mediated by Ca2 + Kv1.3 and KCa3.1 ion channels.
  • Lack of functional Ca2 + channels inhibits the development of regulatory T cells.
  • Blocking the KCa3.1 ion channels increases cytotoxicity and lysis of tumor cells by NK cells.

For all these reasons, the network of ion channels present in immune cells that regulate both innate and adaptive immune responses are highly interesting targets in the development of new therapeutic tools against cancer.

Tumor Processes And Ion Channels

Some of the tumor processes in which the active participation of ion channels has been described are the following:

  • Cell cycle progression and proliferation

Ionic transport is an integral part of the regulation of cell cycle progression and proliferation.

  • Apoptosis

Ionic fluxes are also crucial in the regulation of programmed cell death pathways.

  • Metabolism of tumor cells and tumor microenvironment

The cellular microenvironment usually presents different physical-chemical characteristics than most normal tissues and contains different types of cells in addition to tumor cells. Ionic channels are essential in the interaction between tumor cells and their microenvironment.

  • Tumor cell motility and invasiveness

The motility and invasion of tumor cells, the main steps in metastasis, require the functional integration of several processes, almost all of them regulated by ion channels.

Antibodies For Ion Channel Research

Specific antibodies against ion channels are a fundamental research tool for the study of cancerous phenomena of proliferation, migration and metastasis, angiogenesis and resistance to apoptosis.

Here we leave you a selection of antibodies for the investigation of ion channels (IC)  from the Genetex company .

1.- PROLIFERATION

C. I.AntibodiesApplicationsReference
Shaker-like K + channelsKv1.1 antibodyICC / IF, WBGTX54875
Kv1.5 antibodyICC / IF, IHC, IP, WBGTX54837
EAG-related K + channelsKv11.1 antibodyICC / IF, IHC, IP, WBGTX54776
Kv11.1 antibodyICC / IF, IHC, IP, WBGTX54785
Kv11.1 antibodyFACS, ICC / IF, IHC, IP, WBGTX54789
Ca2 + activated K + channelsKCA3.1 / KCNN4 antibodyICC / IF, IHC, IP, WBGTX54786
TRPTRPC6 antibodyFACS, ICC / IF, IHC, IP, WBGTX54757
TRPM7 antibodyICC / IF, IHC, IHC-P, WBGTX54865
TRPM8 antibodyICC / IF, IHC, IHC-Fr, IP, WBGTX54866
P2XP2X7 antibodyICC / IF, IHC, IHC-P, IP, WBGTX54794
SOCsORAl1 antibodyFACS, WBGTX54826

2.- MIGRATION AND METASTASIS

CIAntibodiesApplicationsReference
EAG K + channelsKv10.1 antibodyICC / IF, IHC, WBGTX54788
Kir channelsKir3.1 / GIRK1 antibodyICC / IF, IHC, IP, WBGTX54831
TRPTRPM7 antibodyICC / IF, IHC, IHC-P, WBGTX54865
TRPM8 antibodyICC / IF, IHC, IHC-Fr, IP, WBGTX54866
TRPV1 antibodyICC / IF, IHC, WBGTX54762
P2XP2X7 antibodyICC / IF, IHC, IHC-P, IP, WBGTX54794
SOCsORAl1 antibodyFACS, WBGTX54826

3.- TUMORAL ANGIOGENESIS

CIAntibodiesApplicationsReference
TRPTRPC6 antibodyFACS, ICC / IF, IHC, IP, WBGTX54757
SOCsORAl1 antibodyFACS, WBGTX54826

4.- RESISTANCE TO APOPTOSIS

CIAntibodiesApplicationsReference
TRPTRPA1 antibodyICC / IF, IHC, IP, WBGTX54765
P2XP2X7 antibodyICC / IF, IHC, IHC-P, IP, WBGTX54794
SOCsORAl1 antibodyFACS, WBGTX54826

In addition to GENETEX , other of our represented houses also offer specific antibodies against ion channels . You can check the list of each of them by clicking on the links that we leave you below:

COVALAB (+ info)

  • Antibodies to COVALAB ion channels

LSBIO (+ info)

  • Antibodies to LSBIO ion channels

PROSCI (+ info)

  • Antibodies to PROSCI ion channels

ST. JOHN´S LAB (+ info)

  • Antibodies against ion channels of ST JOHN´s LAB

ORIGIN (+ info)

  • Antibodies to ion channels OriGene