Detection of antibody chains

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Detection of antibody chains

Postby nekameneka » Nov 02 2016 2:52 am

Hi everyone, I have question about detecting antibody heavy chains in WB.
I have done IP with polyclonal rabbit antibody (HRP linked secondary antibody). I don't have problem detecting interacting proteins but I do have second thoughts after detection of originally pulled protein with this rabbit antibody (which I want to stain to have control and conformation it is there), I am not sure is it my protein or is it a heavy chain of the antibody (they are quiet same size). Is there some way to tell?? Should I run an empty sample containing only antibody to make sure? What are my options?
Thanks! :mrgreen:
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Re: Detection of antibody chains

Postby mdfenko » Nov 02 2016 11:15 am

if you used a secondary antibody for the ip then you most likely precipitated igg. secondary antibodies are usually made against igg.
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Re: Detection of antibody chains

Postby relaxin » Nov 02 2016 11:28 am

Yes, you should have a negative control using a mock IP (without sample) to check on possible detection of the antibody H or L chain, if the size of the protein of interest is similar to either chain. Some second antibodies cannot detect IgG chains of certain species. You can do a search.
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Re: Detection of antibody chains

Postby nekameneka » Nov 02 2016 6:22 pm

Sorry I did not mean I did IP with secondary antibody. I did it with primary but stain my WB with primary and secondary HRP linked antibody.
Thank you for the replies. I will do the proper control without any sample.

Edit: I have additional very simple question. I seeded the cells and after 24 hours I transfected them with plasmids I wanted, after 24 hours I changed the medium and stimulated my cells with RNA for 24 hours and than I did lysis. My question is, in case of cells I do not treat with RNA (as a control) can I do the lysis 24 hours after protein transfection or I need to wait for additional 24 hours (time I used for treating stimulated samples)?? Well I know samples and controls should always be treated equally but since I have so many I was wandering is it appropriate to lyse my non-stimulated cells before?
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Re: Detection of antibody chains

Postby relaxin » Nov 03 2016 11:32 am

You should treat the control in the same way as the treated samples. For the control, you add the solvent without RNA (which is water or buffer). As for the handling of too many samples at one time, you can just rinse the cells with PBS and store the plates without any buffer at -80 C until you have time to process all of them.
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Re: Detection of antibody chains

Postby nekameneka » Nov 03 2016 10:42 pm

Thanks a lot for the advice. -80 without any buffer, great, might also be good for more effective lysis.
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Re: Detection of antibody chains

Postby nekameneka » Nov 08 2016 10:36 pm

Hi, I have another question related to plasmids. Most of my plasmids are pEF-Bos but I have one which is pCMV3, is it ok for my experiment to transfect them together? I know it is recommended to always use same plasmids but my question is if this mixing is big no no or not??
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Re: Detection of antibody chains

Postby relaxin » Nov 09 2016 10:26 am

For transient expression, it is fine. For stable expression, it may be wise to use vectors with different antibiotic resistance to ensure the cells harbors both plasmids. pCMV3 vector has neomycin resistance, but I do not find any antibiotic resistance for pEF-Bos vector.
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Re: Detection of antibody chains

Postby nekameneka » Nov 19 2016 5:47 am

Hi, thank you for your reply.
I have another question but I didn't want to make a whole new topic.
I have purified protein and I have some doubts about its concentration. I measured A280 protein concentration on Nanodrop and I also did Bradford assay, but results I got are very much different between those two methods. I understand difference is expected but not such a huge gap. For example Nanodrop says in one sample (it is diluted) I have 0.079 mg/ml but Bradford says I have 1.5 mg/ml in that same sample. My protein buffer does not contain detergents which could interfere with Bradford so I am wondering what is the problem, any ideas?

Thank you very much, I find this forum very helpful. :)
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Re: Detection of antibody chains

Postby mdfenko » Nov 21 2016 7:16 am

absorbance at 280 nm is reading ring structures in the amino acids which make up the protein. if there are fewer than in the average protein then the reading will be low relative to other methods.

also, there may be something in the buffer which interferes with one of the methods you used.

you may also have made an error in your dilution (not necessarily the calculation but maybe mechanically, eg pipet calibration may be off).
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Re: Detection of antibody chains

Postby nekameneka » Nov 21 2016 10:35 pm

Thank you, I still have no idea what is the problem, I also tried higher concentrated protein samples and results is same, gap is huge. My buffer is just usual 50 mM Tris buffer, with 150 mM NaCl, 5 mM MgCl2 and 2 mM DTT. As far as I know and I searched none of these interfere with both methods. My pipettes are fine and calibrated every 6 months, something would be very wrong with pipetting if the difference is so huge and I believe that is not the reason since I repeated several times :cry:
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Re: Detection of antibody chains

Postby mdfenko » Nov 22 2016 7:24 am

are you reading the bradford with the nanodrop? if not, then maybe the nanodrop is out of calibration?

dtt will interfere with the bradford...

you may want to try reading at 215nm. that way you read peptide bonds. run a standard curve using any (cheap) purified protein, in the same buffer as your sample, then compare with your sample.
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Re: Detection of antibody chains

Postby nekameneka » Nov 23 2016 9:13 am

Hi, I don't read Bradford with Nanodrop.
I read it at 595nm, ok, I will try at 215nm.

I have another question, again, not related to my concentration assays. I did SEC experiments with my purified protein and I got 2 peaks making me wonder if it aggregates. Right now I am not able to do MALS but maybe soon. What I did is ultracentrifugation (100.000 rpm for 10mins) and I found pretty similar concentration of my protein in total and in supernatant while there was almost nothing in pellet by Nanodrop and by SDS-PAGE. It crosses my mind that two peaks in SEC can come from possibility that my protein self-oligomerize so I have earlier peak of dimers and later peak of monomers? Is that possible? Or I most likely have aggregations??

Thanks again! :)
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Re: Detection of antibody chains

Postby mdfenko » Nov 23 2016 2:55 pm

just to be sure, i was suggesting reading at 215 instead of 280, not the bradford.

can you check the calibration of the nanodrop?

did you run standards for sec? if so, then you can estimate the apparent mw of the peaks. that way you can determine if the heavier peak is in multiples (and how many) of the lighter peak.

if it's not in multiples then there could be differences in folding, post translational modifications, or proteolysis.
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Re: Detection of antibody chains

Postby nekameneka » Nov 23 2016 11:48 pm

Thank you!! yes yes, nanodrop at 215nm. I will check the calibration of the nanodrop.
I did not run the standards for SEC, I do plan to do that. :)
Thank you very much for your help!
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