I am currently in the process of propagating primary human macrophages for protein samples to be used in Western Blot.
With cultured cells I have used the RIPA buffer method (RIPA buffer plus protease/phosphatase inhibitor is added to cells on ice, cells are incubated with the RIPA buffer for 30 mins, mixing every 10 mins. Lysate is sonicated for 10 secs on ice, avoiding foaming. Lysate is centrifuged at 12,000 x RPM for 5 mins and stored at -20).
However, I wanted to know whether I should denature the proteins before storing by boiling the sample at 95 for 5 mins? Or is this only when using an SDS lysis buffer?
No -- as long as you store samples at -80oC you should be fine but you should aliquot your master sample and thaw out aliquots for gels as required. Also, always keep samples on ice until they have been boiled in SDS sample buffer. NB: Some enzymes are remarkably active even in RIPA lysis buffer....
Hi, usually what I do (especially for "sensitive" sample, i.e if I have to evaluate phosphorilation levels or similar) is to aliquot my protein lysate, done in RIPA, as you have done, in "loading quantity", add 5X laemmli buffer (to 1X), boil it and then store at -20. If I have no time for doing it, I store the whole amount of lysate at -80 for maximum 3,4 days..I have found that sometimes also in -80 there are some change in proteins levels (especially phosph-)
So to answer you...yes you should ! :) hope this helps
Thank you Fulvio. I have seen this recommended in online protocols. Do you know if Laemmli buffer can be used with Invitrogen NuPAGE® Novex 10% Bis-Tris Gels?
Hi Jose, you're welcome Yes I have used laemmli buffer in precast invitrogen NuPage gels. they recommend to use their loading bf because it contains Lithium Dodecyl Sulfathe instead of Sodium DS and it is more stable...so what I have done is running at lower voltage (slower run) (150V). The bands were sharp and ok. remember to put DTT or BME in loading bf..enjoy!
Hello Jose, denaturation may not be necessary; I normally snap-freeze my samples in liquid nitrogen before storage at -80 degree. Your proteins should retain their stability under such treatment conditions. I only add laemmli buffer to my samples if I want to store at -20 degree. Best wishes.
Thanks Samantha and Benedict. I will do an optimisation to determine whether snap freezing or boiling is better for protein preservation. Thanks for your help :)
In our experience, it was not necessary to denature the proteins prior to storage in RIPA buffer. We always denatured at the time that we ran the Western.
I agree with Carol. We always freeze our sample without denaturing them and boil the amount we are going to load only. If you freeze the samples after denaturing, they will coagulate and precipitate.
I too generally freeze my samples and only denature them before running them out. Although, denaturing them prior to freezing won't hurt your samples.
Hi Jose, Boiling with reducing agents such as DTT or 2-ME, help changing the protein to linear form, necessary before doig SDS-PAGE. it is not nessary to boil your sample before storage. So if u boil your sample and then store it, it is necessary you boil it again before doing SDS-PAGE, but this time in a short period. 3 min boiling is enough. Good Luck
Thanks for all of your advice on this one. Since I use NUPAGE gels, I will mix my samples with LDS and DTT and boil them before storing at -80. I will then boil them again before electrophoresis. All of your comments are much appreciated!
When collecting samples on ice, I have snap frozen the samples immediately without boiling. Then boil at 100 degrees for 5 mins before loading onto SDS-PAGE has worked really well for me.
Once you put your sample into the loading buffer (for protein) you can directly store them without any boiling. Of course the boiling is gonna be necessary for the SDS-PAGE analysis right before running the gel. After the boiling I suggest a centrifugation step for 30 sec 1 min at top speed at 4C to avoid all the precipitates (protein aggregation, DNA...)
Jose, just a word of caution..if you use LDS loading ...there's no need to boil (i.e 100°C ) the sample, but just 5min @70°C. LDS is heat-sensitive. BUT if you use SDS-loading, then 5min@100°C is your cup of tea...enjoy and good luck
No -- as long as you store samples at -80oC you should be fine but you should aliquot your master sample and thaw out aliquots for gels as required. Also, always keep samples on ice until they have been boiled in SDS sample buffer. NB: Some enzymes are remarkably active even in RIPA lysis buffer....
hi guys
i saw here is a suitable place to ask u sth. i always denaturate in LDS then heat them 70 degree 10 min and keep them at -20 it is a really good method for keeping samples before running them.What problem arises if accidentally proteins kept 30min in 70c?aggregation or inactivation of mercaptho?
Hi everyone, I have a question: what would happen if the sample was heated in 95C for 5 minutes without 4xLDS but the 4xLDS was added afterwards?
Vanessa - you might not get proper denaturation, SDSprotein association and, perhaps most importantly, reduction of disulphide bonds if you do not boil in the L/SDS sample buffer.
Hi everyone !
Thanks for the nice discussion, i use RIPA buffer to collect the protein lysates and without undergoing centrifugation directly store the cell lysates at -80 or -20oc depends on my storage time.When i need to run the SDS i just centrifuge the cell lysate at 13500 for 10 mints and heat to denature the protein supernanet at 95oc for 10 minutes. The results seems quite nice, Is it right way to store the cell lysate directly or centrifuge first and store only protein supertanent?
How long is better to store at both 20oc and -80oc ?
But if any further suggestions to modify the protocol to make it much better so as to avoid any unspecific bands are welcome.
With Regards.
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