12 replies to this topic

[sueanne]

Hey guys!

I'm new here. I wanted to determine concentration of my antibody. do you guys think i can use nanodrop and measure the absorbance at 280nm. if i use BCA assay, can i use BSA as my protein standard or should i use bovine gamma globulin as my standard. my budget is limited so i was thinking to use BSA. is it ok?

[Gerard]

Antibody's are mostly IgG's, ask a clinical chemistry lab to measure IgG content.

[klinmed]

sueanne, on Feb 27 2009, 06:46 AM, said:

Hey guys!

I'm new here. I wanted to determine concentration of my antibody. do you guys think i can use nanodrop and measure the absorbance at 280nm. if i use BCA assay, can i use BSA as my protein standard or should i use bovine gamma globulin as my standard. my budget is limited so i was thinking to use BSA. is it ok?

I usually use absorbance at 280 nm to quantitate immunoglobulins provided they are pure (for example free of carrier protein). Most antibodies have a 1% extinction coefficient of 13-15. A 1 mg/ml solution of a IgG can be expected to give an absorbance of about 1.36 in a 1 cm light path cuvette. A similar value can be used with the nanodrop equipment. Thus absorbance at 280/1.36 = mg/ml. If your Ab has a high concentration you may need to make a dilution before reading the absorbance.
Determination of Ab concentration by spectrophotometry is standard practice in immunology. If you are interested, you can find lists of extinction coefficients in standard text books.

Hope this helps.

Edited by klinmed, 02 March 2009 - 11:52 AM.

[bachai]

sueanne, on Feb 27 2009, 03:16 PM, said:

Hey guys!

I'm new here. I wanted to determine concentration of my antibody. do you guys think i can use nanodrop and measure the absorbance at 280nm. if i use BCA assay, can i use BSA as my protein standard or should i use bovine gamma globulin as my standard. my budget is limited so i was thinking to use BSA. is it ok?

If your antibody is a purified Ig prep, nanodrop on BCA, or even OD280 (which eats nearly nothing of your budget but is much less sensitive) would be alright. If you need accuracy of course bovine IgG is a better standard than BSA but if you need rather a relative estimation of Ig concentration from one Ig prep to another BSA would be fine.
If your antibody prep contains other proteins you may need to send your sample to a clinical chemistry lab for specific IgG quantitation, e.g. by immuno-nephlometry.
Good luck!

[manuelo]

Hi,

I have a question regarding IgG. When running the abs on a protein gel, the heavy chain gives a strong band while the light chain only gives a weak band. Is this normal?

Second, I have used the protein chips from Agilent to use the Bioanalyzer (Agilent) to get an idea of the purity as well as concentration. Also here, the heavy chains constitutes approx 80% of the total amount of IgG while the light chains only approx 16%. I'm just getting confused here, shouldn't there be a 1:1 ratio or am I missing something obvious?

Using the bioanalyzer the heavy chain and the light chains gives separate concentrations (since they are separated and measured individually), for example in one sample 130ug/ml heavy chains and 25ug/ml light chains. Should I then combine the two concentrations, and then get the total IgG concentration?

Best regards,
M

Edited by manuelo, 04 March 2009 - 08:32 AM.

[mdfenko]

manuelo, on Mar 4 2009, 06:41 AM, said:

Hi,

I have a question regarding IgG. When running the abs on a protein gel, the heavy chain gives a strong band while the light chain only gives a weak band. Is this normal?

Second, I have used the protein chips from Agilent to use the Bioanalyzer (Agilent) to get an idea of the purity as well as concentration. Also here, the heavy chains constitutes approx 80% of the total amount of IgG while the light chains only approx 16%. I'm just getting confused here, shouldn't there be a 1:1 ratio or am I missing something obvious?

Using the bioanalyzer the heavy chain and the light chains gives separate concentrations (since they are separated and measured individually), for example in one sample 130ug/ml heavy chains and 25ug/ml light chains. Should I then combine the two concentrations, and then get the total IgG concentration?

Best regards,
M

the molar ratio may be 1:1 but the actual amount of protein will be different because they are not the same size.

what you found sounds okay and, yes, you can add the totals (not the concentrations) together.

[sueanne]

Hey guys


Thank you so much

[sueanne]

klinmed, on Mar 2 2009, 09:40 AM, said:

sueanne, on Feb 27 2009, 06:46 AM, said:

Hey guys!

I'm new here. I wanted to determine concentration of my antibody. do you guys think i can use nanodrop and measure the absorbance at 280nm. if i use BCA assay, can i use BSA as my protein standard or should i use bovine gamma globulin as my standard. my budget is limited so i was thinking to use BSA. is it ok?

I usually use absorbance at 280 nm to quantitate immunoglobulins provided they are pure (for example free of carrier protein). Most antibodies have a 1% extinction coefficient of 13-15. A 1 mg/ml solution of a IgG can be expected to give an absorbance of about 1.36 in a 1 cm light path cuvette. A similar value can be used with the nanodrop equipment. Thus absorbance at 280/1.36 = mg/ml. If your Ab has a high concentration you may need to make a dilution before reading the absorbance.
Determination of Ab concentration by spectrophotometry is standard practice in immunology. If you are interested, you can find lists of extinction coefficients in standard text books.

Hope this helps.

Thanks so much klinmed. this is my first time dealing with antibodies and i have no experience in immunology. still trying to learn

[Nora]

sueanne, on Feb 26 2009, 10:46 PM, said:

Hey guys!

I'm new here. I wanted to determine concentration of my antibody. do you guys think i can use nanodrop and measure the absorbance at 280nm. if i use BCA assay, can i use BSA as my protein standard or should i use bovine gamma globulin as my standard. my budget is limited so i was thinking to use BSA. is it ok?

From my experience, Nanodrop is not working reliable with protein 280nm and for BCA, Bradford, etc. you should use cuvettes anyway since the samples are not suitable for small volumes.

If you want to work in small volumes (you can do that with purified proteins like antibodies) you should use a NanoPhotometer from Implen, it is perfect for protein samples since their technology is not depending on surface tension of your samples. BCA, Lowry, etc. is also possible since the instrument works with cuvettes, too.

By the way, BSA is not a good standard for antibodies, since the molar extinction coefficient is far away from your antibody samples. You may want to use the molar extinction coefficient calculated from databases like http://www.expasy.ch/ so you would be able to determine your 280 factor without any standard.

Hope I could help!

[DrMellotron]

The range of extinction coefficients given for immunoglobulins in the literature suggests that measurement of antibody concentration by absorbance is unlikely to be accurate in absolute terms. However, it can be a very reproducible procedure. In my experience it is the best method for ensuring consistency in methods requiring use of antibodies at a given concentration, once the method has been standardized using antibodies measured in that way. Reproducibility between antibodies of the same source and purity should be good, but will decrease with increasing difference in form (e.g. source, preparation, species). Values are even less likely to be accurate in absolute terms for monoclonal antibodies, although reproducibilty of measurement with the same clone should be good.
Absorbance is only accurate for pure immunogloblins. For impure preparations an assay such as radial immunodiffusion is ideal. It is simple to run and a range of species specific assays are commercially available.
Measuring the same antibody by different methods often does not give the same value, unless you use a include a standard in them all.

[shane]

If you desire to work in little volumes you should use a NanoPhotometer from Implen, it is flawless for protein trials since their expertise is not counting on exterior stress of your samples. BCA, Lowry, etc. is furthermore likely since the equipment works with corvettes, aslo..

[dietacai]

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[sbyrne27]

Hi

You might also take into account that the IgG bands might not always so up at there usual spots on the gel. As some antibody can be aggregated, fragmented and so on

cheers

sbyrne27

mdfenko, on 04 March 2009 - 08:35 AM, said:

manuelo, on Mar 4 2009, 06:41 AM, said:

Hi,

I have a question regarding IgG. When running the abs on a protein gel, the heavy chain gives a strong band while the light chain only gives a weak band. Is this normal?

Second, I have used the protein chips from Agilent to use the Bioanalyzer (Agilent) to get an idea of the purity as well as concentration. Also here, the heavy chains constitutes approx 80% of the total amount of IgG while the light chains only approx 16%. I'm just getting confused here, shouldn't there be a 1:1 ratio or am I missing something obvious?

Using the bioanalyzer the heavy chain and the light chains gives separate concentrations (since they are separated and measured individually), for example in one sample 130ug/ml heavy chains and 25ug/ml light chains. Should I then combine the two concentrations, and then get the total IgG concentration?

Best regards,
M

the molar ratio may be 1:1 but the actual amount of protein will be different because they are not the same size.

what you found sounds okay and, yes, you can add the totals (not the concentrations) together.