Heat-induced antigen retrieval utilizing modified Tris-EDTA buffer for reprobing of Western blots on nitrocellulose paper.
Anal Biochem
; 672: 115179, 2023 07 01.
Article
in En
| MEDLINE
| ID: mdl-37150424
ABSTRACT
The development of heat-induced antigen retrieval technologies with Tris-EDTA buffer has dramatically improved immunostaining of specific antigens for routine immunohistochemical detection (Krenacs et al., 2010) [1]. However, little evidence exists on whether heat-Induced antigen retrieval utilizing Tris-EDTA buffer can strip western blot (WB) membranes and allow sequential reprobing. Here, we serendipitously discover that â¼95 °C Tris-EDTA buffer with 0.01% Tween 20 could repeatedly strip the Nitrocellulose membranes (NC). After electroblotting, NC blots were soaked into Tris-EDTA stripping buffer (â¼95 °C, 10-25min) and we could perform at least five rounds (the following antibodies used Vinculin, Atg7, Caspase-3, UBA5, JNK and ERK1/2) stripping in sequential chemiluminescent detections. The NC membranes also show clear western signals and background without losing transferred proteins during the reprobing process of WB. Hence, this study report additional new roles of the heat-Induced antigen retrieval Tris-EDTA buffer with 0.01% Tween 20. The method is simpler, more affordable and harmless for the nitrocellulose paper, which will be helpful for effective reprobing in western blotting applications.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Tromethamine
/
Hot Temperature
Language:
En
Journal:
Anal Biochem
Year:
2023
Document type:
Article