Activation Reagent

Instruction manual

FOR IN VITRO AND RESEARCH USE ONLY
NOT FOR USE IN CLINICAL DIAGNOSTIC PROCEDURES

First Edition (Revised on April, 2016)

PRODUCT INFORMATION

TGF-βs are secreted by several type of cells. All three TGF-βs are synthesized as disulfide-linked homodimers containing a propeptide region. After being synthesized, the TGF-β homodimer interacts with a Latency Associated Peptide (LAP, an N-terminal signal peptide of 20-30 amino acids derived from the TGF beta gene product), forming a complex called Small Latent Complex (SLC). This complex remains in the cell until it is bound by another protein called Latent TGF-β-Binding Protein (LTBP), forming a larger complex called Large Latent Complex (LLC). It is LLC which is secreted to the extracellular matrix (ECM). That is to say, most TFG-β is secreted as a latent form(LLC) in biological samples. The sample needs to be further processed in order to release TGF-β from LLC. After activation, the TGF-β level in samples can be accurately measured since the antibodies in the ELISA kits for TGF-βs is specifically against to the free TGF-β.

REAGENT

REAGENTQuantity
Activation Reagent A20mL (1×)
Activation Reagent B20mL (1×)

EXPECTED APPLICATIONS

It’s applicable for TGF-β release from serum/plasma/cell culture supernates samples, et al.. After activation, the TGF-β level in samples can be accurately measured since the antibodies in the ELISA kits for TGF-βs is specifically against to the free TGF-β.

STORAGE AND PERIOD OF VALIDITY

Stored at room temperature for one months.

SAMPLE ACTIVATION PROCEDURE

Serum/Plasma
1.To 50μL of serum/plasma, add 10μL of Activation Reagent A. Mix well.
2.Incubate 10 minutes at room temperature.
3.Neutralize the acidified sample by adding 10μL of Activation Reagent B. Mix well. Add 80μL Standard Diluent.
Mix well. Assay immediately.
4.The concentration of samples must be multiplied by dilution factor, 3.Cell culture supernates
1.To 100μL of cell culture supernate, add 20μL of Activation Reagent A. Mix well.
2.Incubate 10 minutes at room temperature.
3.Neutralize the acidified sample by adding 20μL of Activation Reagent B. Mix well. Assay immediately.
4.The concentration of samples must be multiplied by the dilution factor, 1.4.

IMPORTANT NOTES

1. Ensure that pH of samples after neutralization is within 7.2-7.6. Adjust the volume and corresponding dilution factor of the neutralization reagent as needed.
2. Activated samples must be assayed immediately. Do not freeze activated samples.
3. The solutions may be stored in polypropylene bottles at room temperature, and they should be screwed tightly to protect from evaporation.
4. Wear protective clothing and safety glasses during preparation or use of these reagents..
5. Do not use these reagents for activation of standards from the kit.