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The Bcl-xL (54H6) Rabbit mAb #2764 recognizes a portion of the human Bcl-xL sequence which is retained in all three isoforms of Bcl-xL. Please see UniProt for the precise isoform sequences:
https://www.uniprot.org/uniprot/Q07817#sequences
The doublet is most likely Bcl-xL and Bcl-xB, as Bcl-xS has a lower predicted molecular weight (19 kDa versus 25-26 kDa).
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We do not routinely validate our products for use in fluorescent western blot; however, many are expected to work well in this application with a few protocol modifications. When performing fluorescent western blot, we recommend omitting Tween-20 from the blocking step as well as allowing the membrane to dry completely prior to imaging to achieve optimal results. Our recommended protocol for fluorescent western blotting can be found here.
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We have not tried counterstaining in-house, however, the paper below mentions using Hoechst as a fluorescent DNA dye to quantify the cells:
Baker, D. J. et al. (2004) BubR1 insufficiency causes early onset of aging-associated phenotypes and infertility in mice. Nat. Genetics 36, 744-749
You may be able to find other references on the web, as this kit is popular and there are many publications citing its use. We use hematoxylin routinely in our IHC experiments, and, consequently, have limited experience with alternatives.
For further suggestions, it may be helpful to seek out information within the Histonet (http://www.histosearch.com/histonet.html). This is a well-monitored discussion forum for histology professionals. There is an archive of queries that can be searched, or a new query can be posted. There may be suggestions or observations using these methods that could prove helpful.
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The suggested protocol for treating cells with poly(dA:dT) can be found below:
Poly(dA:dT) Treatment Protocol (5 μg/mL final concentration)
Reagents:
- Poly(deoxyadenylic-thymidylic) acid sodium salt (CST product: Poly(dA:dT) Sodium Salt #47945)
- FuGENE 6 Transfection Reagent from FuGENE
Procedure:
24 hours prior to transfection:- Plate cells in two 15 cm plates with 21 mL media with serum (no antibiotic)
On transfection day:
- Prepare two falcon tubes per plate to be transfected:
- For mock: One tube labeled “FuGENE” and one tube labeled “Mock”
- For poly(dA:dT): One tube labeled “FuGENE” and one tube labeled “Poly(dA:dT)”
- Add 4.5 mL of 1X serum-free medium to ea…