how to start for effective RNAi screening--scale up question - (Feb/17/2006 )

I have three RNAi constructs ready for each gene of my interest, which are made with Invitrogen Block-it miR RNAi kit. I have done Hela cell transfection before for subcellular localization. But I have no experience with protein work before. I have several questions that are confusing me:

1. How many cells I should start with so that I would have enough cells for Western analysis later? Can I use 6-well plate or better with 100 mm dish?

2. I want to kill those untransfected cells with selection so that it will give me better idea which miR RNAi is really working. When should I add the antibiotics? one or two days post transfection?

3. To make protein samples for Western analysis, what time point is better? 1 or 2 days after selection?

4. Any particular cell lysis buffer I should use for protein isolation?

I'm using long-Hela cell, which has several times less cells when confluent compared to the regular Hela cells.

Thanks for your help. This is urgent as my boss don't want me spend too much time on preparing.

hi
using "normal" HeLa cells in 6well plates, i can do 3-4 western blots with 20µg proteins for one. So starting with 6well plate would be ok for you.
i think that in general cases, antibiotic should be added 2days after transfection. but if your transfection is thursday morning, friday at quite end of day (let's say 19h) is ok if you don't want to come back during we...
1 or 2 days post transfection is a relevant question. If you don't know and don't have enough material, 48h is the best choice for 1st try. But better is to do 24 48 and 96h post transfection.

for cell lysis, i uses
i use this buffer for my cell lysis...

NaCl 150mM (3ml NaCl 1M)
EDTA pH8 2mM (160µl EDTA 250mM)
NP40 1% (2ml NP40 10%)
Tris HCl 50 mm pH 7.5
ddH2O qsp 20ml

maybe you can try it?

you may also have a look at hes threads :
http://www.protocol-online.org/forums/inde...wtopic=5090&hl=

fred

Thank you so much, Fred. Is there anybody here having experience with long-Hela cells? I'm using blasticidin for selection, which is very potent. I found that it kills most of sensitive cells after 2-day treatment at concentration as low as 4 ug/ml. If my transfection efficiency is not high enough, then I may not have enough cells left for protein isolation if I use 6-well plates. Is that right?