Nowadays, we still find antibodies at the core of many key lab techniques, but there’s a big difference in what’s going on downstream of the IP today. A few years ago researchers were looking at varying shades of grey on a gel; a gel that didn’t eat up too much grant money. Now, the downstream scenario is a bit different.
First, open up that wallet because downstream analysis doesn’t come cheap when using ChIP-Seq, or ChIP on Chip. Analysis? Yeah, there’s a lot more of that too. The old adage of garbage in/garbage out not only means time and effort wasted……….choosing a non-functioning antibody nowadays can create some very expensive trash. How do you know which antibodies to trust? Should you click around online until something grips you, or maybe take advantage of that 3 for 1 promo on that annoying Flash banner ad that keeps reloading? It’s a big decision, especially because one wrong move here can sink your experiments later on. Our hands are jittery just thinking about it; of course that might just be the caffeine.??
Quality Control: Failure is Your Friend
Most researchers go with an antibody supplier rather than producing their own. In the ultra-fast pace of research nowadays, who can resist adding antibodies against H3K27me3 to that checkout cart and presto! No wasted time injecting rabbits and waiting for bleeds. No waiting for cell lines to express the right antibodies. Nope, you can get right down to business and bang out those Nobel-prize winning experiments. But convenience comes with concessions. As Jim Bone at antibody provider Active Motif (Carlsbad, CA) points out, “There are pros and cons to both options. Generating antibodies solo is very time-consuming and labor-intensive. However, you can really get your hands in there, designing the immunogen yourself and performing specific validation tests to make sure the antibodies work the way you want them to.”?
Antibody suppliers handle all the heavy lifting for you, but they have a pretty tough job considering the diversity of downstream uses for antibodies. “You really have to trust your supplier,” cautions Bone. “The antibodies may not be exactly what you want. They’re not tailored specifically for your experiment.” For example, some commercially available antibodies might work well for westerns, but not for ChIP.
Fail Early, Succeed Often?
To see exactly how these binders will work in real-world applications, suppliers typically run their antibodies through validation tests and weed out the non-performers, but it can vary a bit from supplier to supplier. How much weeding out happens before that tube ends up on your lab bench? It varies but we learned Active Motif fails up to 50% of their antibody batches during specificity and validation tests! Seem like a lot? It did to us, but better them than you right?, particularly when many of the QCs are functional (i.e. laborious). In general, there are no particular antibody characteristics that scream out, “I’m good for westerns” or “I’m good for IPs”, so, Bone says, you really just have to try them out once they are produced. Bone filled us in on the QC lifecycle of a typical antibody at Active Motif: ???
- Specificity Screening: Checking for high titer and running dot-blot tests for specificity lets them quickly weed out bad antibody batches. Many of their antibodies are modification-specific, so specificity testing includes asking whether an antibody recognizes, say, a phosphorylated form of Protein X, but not the unphosphorylated form. ???
- Western Blots: Antibodies that look good at this stage are probably going to be a hit. “If you see good titer (around 1 : 5,000), and it’s really specific and clean on a Western blot, then odds are it’s probably going to work in ChIP…but you never know until you try it,” explained Bone.???
- ChIP and IF Testing: Because it is focused on the epigenetics, transcription factor, and chromatin fields, Active Motif validates its antibodies with westerns, immunofluorescence (IF) assays, and chromatin IPs before they release them for sale, so customers know up front what experiments each antibody will work in.? ??
How Fit is Your Antibody??
Good question…and probably not one you want to be asking during data analysis. Flex your consumer muscles, and don’t be afraid to ask your supplier upfront. Get whatever info they have for the exact lot you’re ordering. Make sure each batch has its own unique lot number too. Remember, you’re not just paying for what arrives in that tube. QC is part of the price you’re paying for your product!? ?
Thanks to Jim Bone at Active Motif for filling us in. You can find more details on their QC processes and a cornucopia of antibodies, atwww.activemotif.com