我应该使用哪种固定剂来固定自己的细胞或组织,从而进行抗体标记?
醛类固定剂(例如甲醛、戊二醛)可与各种细胞成分交联,这有助于保持蛋白质和细胞的形态。但一些抗原可能会因交联而被掩藏,需要通过抗原修复的方式暴露掩藏的抗原结合位点。此外,醛类固定剂需要通过通透处理使抗体进入细胞。甲醇和丙酮等有机溶剂可凝聚蛋白质并通透细胞,但细胞形态会受到破坏。表面抗原无需固定即可标记。
Where do I find the expiry date of Pierce 16% Formaldehyde (w/v), Methanol-free (Cat. No. 28906)?
We guarantee the functionality of Pierce 16% Formaldehyde (w/v), Methanol-free for one year from date of shipment, if stored correctly. See Limited Warranties for consumables under the General Terms and Conditions of Sale.
Find additional tips, troubleshooting help, and resources within our Labeling Chemistry Support Center
What are the options for crosslinking proteins to nucleic acids, and what are the differences between them?
We offer the following crosslinkers for protein to nucleic acids: formaldehyde (Cat. No. 28906), SPB (Cat. No. 23013), SDAD (Cat. No. 26169), sulfo-SDAD (Cat. No. 26175), and sulfo-SBED (Cat. No. A39260).
Formaldehyde can be used for DNA-DNA crosslinking as well as DNA-protein crosslinking. While it is good for crosslinking proteins that are in direct contact with DNA, formaldehyde cannot link proteins that may be bound in a complex with other proteins but are not in direct contact with DNA. EGS or DSG can crosslink proteins to other proteins that are in direct contact with DNA and crosslinked to DNA by formaldehyde.
SPB, Sulfo-SDAD, and Sulfo-SBED are all photoreactive crosslinkers. SPB is an NHS-ester and psoralen heterobifunctional crosslinker that conjugates primary amines on proteins to DNA via photo-activated intercalation of psoralen to pyrimidine bases. The psoralen tricyclic planar ring system intercalates into double-stranded, and to a lesser extent, single-stranded DNA and RNA. The photoreactive psoralen group provides more selective covalent binding to nucleic acids than either phenyl azide or diazerine linkers, which makes SPB the best choice. The diazerine on sulfo-SDAD and the phenyl azide on sulfo-SBED are not selective for nucleic acids and can crosslink other biomolecules in the same besides any protein-DNA interactions. Sulfo-SBED involves biotin transfer rather than actual linking.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.
What fixative should I use to fix my cells or tissues for antibody labeling?
Aldehyde-based fixatives (e.g., formaldehyde, glutaraldehyde) crosslink various cellular components, which helps retain proteins and cell morphology, but some antigens can be masked by the crosslinking, requiring antigen retrieval methods to unmask the antigen binding sites. Also, aldehyde-based fixation requires permeabilization to allow entry of antibodies. Organic solvents such as methanol and acetone condense proteins and permeabilize the cells, but cell morphology can be compromised. Surface antigens can be labeled without fixation.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
What is the difference between formalin and formaldehyde?
The terms formalin and formaldehyde are often used interchangeably, although the chemical composition of each fixative is different. Formalin is made with formaldehyde but the percentage denotes a different formaldehyde concentration than formaldehyde solutions. For example, 10% neutral-buffered formalin (NBF or simply formalin) is really a 4% formaldehyde solution; the basis for this difference is that historically, formalin was prepared with commercial-grade stock formaldehyde, which is 37 to 40% formaldehyde, by diluting it 1:10 in phosphate buffer.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.