I read about boster ELISA kit rat by Boster immunoleader ( I give attachment)
In Chapter Preparation at the Point 2
2. Activate the sample ( if I want to analyze the active form)
• Cell culture supernate, urine: add activating reagent pro rata, i.e. add 20μl of Solution A into 100μl of sample, 10 min later, add 20μl of Solution B. PH 7.0-7.6.
• Serum, plasma(EDTA): add activating reagent pro rata, i.e. add 20μl of Solution A into 40μl of sample, 10 min later, add 20μl of Solution B. PH 7.0-7.6.
• It is unnecessary to activate the recombinant TGFβ 1.
• Sample was diluted partly after adding activating reagent, so please pay attention to this when you calculate target protein concentration.
Questions:
If sample is not activated what will we get? Total Latent TGF beta 1?
Total TGF beta 1 can I conclude that are inactive plus active TGF beta 1?
TGFb in a biological sample will generally be present in one of two forms, either as LAP+TGFb (=latent TGFb) or free TGFb. The ELISA only recognizes free TGFb. Therefore, if you do not activate your sample, you will only be measuring the free TGFb in the sample. If you run the assay twice, one without activation (=free TGFb) and once with activation (=free TGFb + latent TGFb), the difference in TGFb values from the two assays will be the amount of latent TGFb in the sample.
TGFb in a biological sample will generally be present in one of two forms, either as LAP+TGFb (=latent TGFb) or free TGFb. The ELISA only recognizes free TGFb. Therefore, if you do not activate your sample, you will only be measuring the free TGFb in the sample. If you run the assay twice, one without activation (=free TGFb) and once with activation (=free TGFb + latent TGFb), the difference in TGFb values from the two assays will be the amount of latent TGFb in the sample.
Free TGFb plays the biological function of TGFb, but its concentration is very low in blood (low pg/ml) and is usually beyond the detection limit of most commercial assay products. BioLegend sells separate ELISA products for Free Active TGFb1, Total TGFb1, and LAP. The Free Active kit is the only kit available on the market.
Depending on the samples origin (cell culture supernatant or serum/plasma) and model, the relevance of every state could be different. The ratio of Latent/active (TGFb activation) also indicates or suggests inflammatory, oxidative or profibrotic state. Take into account that cell supernatant could contain cross-reactive free and latent TGFbeta from bovine serum supplementation (controls required). Answering your questions...
If sample is not activated what will we get? Total Latent TGF beta 1? If you don´t activate the sample you will get only active TGFbeta
Total TGF beta 1 can I conclude that are inactive plus active TGF beta 1? Yes total TGFb= active + latent
The best option, as mentioned, is running both activated and non activated sample, then you will get more information and the ratio. It is true that blood from heatlhy humans or animals will content very low levels or active TGFbeta but these leves could be significantly increase by chronic inflammation, fibrosis, carcinogenesis or acute damage context. Anyway do no forget that in tha case of TGFbeta activity if results are no conclusive (due to quick activation and degradation processes) other option could be to measure Smad2 or/and 3 phosphorylation in response to active TGFbeta presence.
How about LAP+TGF beta+LTBP, it is including in LAP+TGF beta in detection with ELISA or not?
In sample from tissue aorta rat?what we will get if without activation?
Are free TGF beta = active TGF beta?
I attach picture the questions. Thanks Mr. Bruce Patt, Mr Shaoquan Ji , Mr Javier Dotor for your answer
Picture From Cardiovascular Research 74 (2007) 196–206
Ruiz-Ortega et al. (2007) TGF-β signaling in vascular fibrosis
How if the active tgf beta 1 levels in ELISA increase but collagen decrease and vascular smooth muscle decrease too? do you have reason?
I would like to ask a question: How many kDa is TGF-beta?
We used anti TGF-beta1 anti-body to detect TGF-beta1 in human skin cells and find out that TGF-beta1 was expressed at 44kDa and 25kDa(44kDa is almost 2 times lighter than 25kDa). We also used isolated total protein from injured mouse skin to detect TGF-beta1 and find out that TGF-beta1 was mainly expressed at 12.5kDa. Its known that 44 kDa is pro-TGF-beta while 25kDa and 12.5kDa are mature TGF-beta (25kDa is activated dimer). So, is that possible to hypothesize that ECM (extracellular matrix) was included in mouse skin sample, so 12.5 kDa was mainly expressed ?
How many kDa of TGF-beta1 should be expressed in normal human cells?
Thanks so much.
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