How to prepare 0.2% Saponin for lysis of THP-1 derived macrophages?

Hello I'm new to intracellular FACS staining of cells and I'm now trying the saponin permeabilization but have no idea of what saponin product should I choose to order. The permeabilization buffer, according to a published paper that did the same thing with me, is composed of PBS+ 0.1% BSA+ 0.05% saponin, but they didn't show the catlog number of the saponin...Can anyone who has done saponin permeabilization give me some suggestions about either the product or the experiment? Thanks a lot!

I am trying to stain organoids and where I am getting results with different staining protocols, I do noticed difference between protocols, especially when it comes to permeabilization component. Some seem to prefer Saponin and Others Triton-X100, or even both.

I am trying to find a nice paper or some more clear information about why I would consider using the different permeabilization components. Furthermore, I was wondering maybe that for organoids, that normally need longer permeabilization steps a component like Saponin might be more benificially seeing it does not effect protein structure.

Any information about this topic and papers I could read that could give me a more clear answer would be appreciated.

Hi, everyone

I was reading many PCR protocols where I found that 100 pmol = 100uM. I am unable to understand this conversion as I found in google that 1pmol = 1000000 micromol.

can anyone guide me in a detailed way?

Thank you

I'm going to perform a western blot analysis on cell lysate from human

adipose derived mesenchymal stem cells. How much material (number of

cells/protein) is required? Do you usually run your WB samples in

duplicates?

I have a very limited number of cells..

Hello everyone, I am currently conducting my research on identifying bacteria directly from blood culture bottles using MALDI-TOF MS. For direct ID, I am planning to use a chemical solution that is 5% saponin lysis buffer. Could anyone help in providing me with a detailed preparation protocol for it?

We are using pET-32 Xa/LIC kit (Novagen 70072-3)  for cloning. In kit manual there is no media mentioned for plating the transformants. We have used the LB culture medium. But there is no colony found of positive control insert on LB plates. Should we use SOC media for plating?

Is there any difference between LB or SOC media to select transformants?

I am performing differential gene expression analysis and I am not sure what is an ideal cutoff or threshold for log2(Fold change). To my understanding, we only require acceptable cutoff for p-value, however, is there any criteria to decide threshold for fold change?

Helly, I've currently bought 1 mg of PMA from sigma to treat U937 cell line differentiation, but now, I'm a bit confused with how to prepare it.

In the sigma website has written that I need to dissolve PMA to 20 mM concentration, but in term of usage. I need 25-100 ng/ml concentration for optimization which dissolving PMA to 20 mM make me confuse how to properly prepare it.

I've seen the suggestion that just dissolve 1 mg or 5 mg of PMA with 1 ml of DMSO and gradually dilute to required concentration which make me even more confused.

I'm currently setting up immunostaining of prepared slides. The protocol usually is: Let primary antibody incubate at 4C overnight, then the next day add the secondary body on for 2 hours at RT. I started my primary incubation, but something came up tomorrow so I will be gone part of the day. So I could either let the primary go for the rest of the morning and come back in the afternoon to put the secondary on for 2 hours, or take the primary off in the morning, put the secondary on but leave it for like 4-5 hours RT. Is one better to do than the other?