Overview

  • Product name
    Caspase-3 Assay Kit (Colorimetric)
    See all Caspase-3 kits
  • Detection method
    Colorimetric
  • Sample type
    Cell Lysate
  • Assay type
    Enzyme activity
  • Assay time
    2h 00m
  • Product overview

    Caspase-3 Assay Kit (Colorimetric) ab39401 provides a simple and convenient means for assaying the activity of caspases that recognize the sequence DEVD.


    The Caspase-3 assay protocol is based on the formation of the chromophore p-nitroaniline (p-NA) by cleavage from the labeled substrate DEVD-pNA. The p-NA can be quantified using a spectrophotometer or a microtiter plate reader reading absorbance at 400 or 405 nm.


    Comparison of the absorbance of p-NA from an apoptotic sample with an uninduced control allows determination of the fold increase in Caspase-3 activity.


    Caspase-3 assay protocol summary:
    - add samples to wells
    - add reaction buffer and DEVD-p-NA substrate and incubate for 60-120 min at 37ºC
    - analyze with microplate reader

  • Notes

    Due to the nature of the substrate, this assay also detects caspase-7 activity.

    Activation of ICE-family proteases/caspases initiates apoptosis in mammalian cells.

     

     

  • Platform
    Microplate reader

Properties

  • Storage instructions
    Store at -20°C. Please refer to protocols.
  • Components 100 tests
    2X Reaction Buffer 4 x 2ml
    Cell Lysis Buffer 1 x 100ml
    DEVD-pNA 1 x 500µl
    Dilution Buffer 1 x 100ml
    DTT 1 x 400µl
  • Research areas
  • Function
    Involved in the activation cascade of caspases responsible for apoptosis execution. At the onset of apoptosis it proteolytically cleaves poly(ADP-ribose) polymerase (PARP) at a '216-Asp-
    -Gly-217' bond. Cleaves and activates sterol regulatory element binding proteins (SREBPs) between the basic helix-loop-helix leucine zipper domain and the membrane attachment domain. Cleaves and activates caspase-6, -7 and -9. Involved in the cleavage of huntingtin.
  • Tissue specificity
    Highly expressed in lung, spleen, heart, liver and kidney. Moderate levels in brain and skeletal muscle, and low in testis. Also found in many cell lines, highest expression in cells of the immune system.
  • Sequence similarities
    Belongs to the peptidase C14A family.
  • Post-translational
    modifications
    Cleavage by granzyme B, caspase-6, caspase-8 and caspase-10 generates the two active subunits. Additional processing of the propeptides is likely due to the autocatalytic activity of the activated protease. Active heterodimers between the small subunit of caspase-7 protease and the large subunit of caspase-3 also occur and vice versa.
    S-nitrosylated on its catalytic site cysteine in unstimulated human cell lines and denitrosylated upon activation of the Fas apoptotic pathway, associated with an increase in intracellular caspase activity. Fas therefore activates caspase-3 not only by inducing the cleavage of the caspase zymogen to its active subunits, but also by stimulating the denitrosylation of its active site thiol.
  • Cellular localization
    Cytoplasm.
  • Information by UniProt
  • Alternative names
    • A830040C14Rik
    • Apopain
    • CASP-3
    • CASP3
    • CASP3_HUMAN
    • Casp3a
    • Caspase 3
    • Caspase 3, apoptosis-related cysteine peptidase
    • Caspase 3, apoptosis-related cysteine protease
    • Caspase 3, apoptosis-related cysteine protease a
    • Caspase-3 subunit p12
    • CC3
    • CPP-32
    • CPP32
    • CPP32B
    • Cysteine protease CPP32
    • EC 3.4.22.56
    • LICE
    • mldy
    • OTTHUMP00000165052
    • OTTHUMP00000165053
    • OTTHUMP00000165054
    • PARP cleavage protease
    • Procaspase3
    • Protein Yama
    • SCA 1
    • SCA-1
    • SREBP cleavage activity 1
    • Yama
    see all

Images

  • H9c2 cells were either untreated (control) or treated with 2µg/ml of recombinant lipocalin-2 (Lcn2) for 24h. Cell lysates were assayed for caspase-3 activity (n = 3, *, p<0.05).

    Image obtained from Xu G et al., JBC, 2012 Feb 11;28(7):4808-17

  • Caspase-3 in Jurkat lysates (3.3 x10e6 cells) following 20 hour exposure to 2 uM Camptothecin (ab120115) or 10 ng/mL anti-Fas Ab (MBL).

Protocols

References

This product has been referenced in:
  • Lam TG  et al. New metformin derivative HL156A prevents oral cancer progression by inhibiting the insulin-like growth factor/AKT/mammalian target of rapamycin pathways. Cancer Sci 109:699-709 (2018). Read more (PubMed: 29285837) »
  • Leger T  et al. Dietary canolol protects the heart against the deleterious effects induced by the association of rapeseed oil, vitamin E and coenzyme Q10 in the context of a high-fat diet. Nutr Metab (Lond) 15:15 (2018). Functional Studies . Read more (PubMed: 29456586) »
See all 63 Publications for this product

Customer reviews and Q&As

1-10 of 12 Abreviews or Q&A

It doesn't work for tissue samples

Inconclusive Good 4/5 (Ease of Use)
Abreviews
We tried to quantify Caspase 3 activity in mouse mammary gland during mammary involution after lactation. We tested different protein amounts fron 50 to 200 microgrames (as indicated in protocol booklet), but the product did not work in any case. We wanted to compare Caspase 3 activity in three samples at different timepoints of involution, and we were not able to observe differences (A), which really exist, as we confirmed by western blot of the same samples (B). We checked the kit was not faulty testig in HC11 mammary cell line, inducing apoptosis with cisplatin 15 ug/ml for 24h (C).
The technical service told us it was a problem with our samples and the did not offer a way to solve our problem.
Then, we found a fluorimetric assay ab39383, which is exactly the same but with a fluorometric substrate instead of a colorimetric one, which is more sensitive as abcam scientific support says in the questions and answer section of the product wall in their website. We aquired the fluorometric substrate and repeated the assay with the same samples. This time, the product worked perfectly (D).
So, for mouse mammary gland tissue samples, I would not recommend this product. It seems it has not enough sensitivity. I would try the fluorimetric assay.

Mr. Adrian Blanco

Verified customer

Submitted Jul 17 2014

Answer

Freezing and thawing may partially degrade caspase activity but the protocol, in step 1.2., suggests it will not be a problem: "Transfer supernatant (cytosolic extract) to a fresh tube and put on ice for immediate assay or aliquot and store at -80°C for future use."

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Answer


ab39401 should recognize all mammalian forms of Caspase 3. One kit is $470, which provides enough solution for one 96 well plate.

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Answer

This kit is good just for cell and tissue lysates but not plasma. If the you are able to collect the platelets from the plasma in a clean manner you will be able to use the kit with the recommended protocol.

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Answer

Thank you for contacting us.

This kit has not been tested with serum sample yet, so we can provide Abtrial discount if customer is interested in testing this kit.

Please note that this product is "for research use only and are not intended for diagnostic or therapeutic purpose.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting us and your interest in our products.

The Caspase 3 Assay Kit (Colorimetric) (ab39401) can be used with tissue samples and we would suggest following the following protocol:

1. Induce apoptosis in tissue (10-100) mg by desired method. Concurrently have control without induction.

2. Re-suspend tissue in 50-400 μl of chilled Cell Lysis Buffer and homogenize using a Dounce homogenizer (10-50 passes) on ice, until efficient lysis is confirmed, by viewing the cells under the microscope.

3. Centrifuge for 1 min in a microcentrifuge (10,000 x g).

Subsequently follow the remaining protocol as described with cell samples.
I hope this information has been of help. If I can be of any further assistance, please do not hesitate to let me know.

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Answer

Thank you for your enquiry.

I am pleased to confirm that this kit (ab39401 Caspase 3 Assay Kit (Colorimetric))is compatible with mouse samples. Here are the references we have from this kit. I hope this will provide some reassurance that this kit is good for a huge array of mammalian samples, including the mouse samples.

I hope this will be helpful. Please do not hesitate to contact me if you have any further questions.


- Anderson, E. J. et. al. Increased propensity for cell death in diabetic human heart is mediated by mitochondrial-dependent pathways. Am J Physiol Heart Circ Physiol, Jan 2011; 300: H118 - H124.

- Argadine HN et al (2009) J Appl Physiol; 107: 438 - 444.

- Barbonetti, A. et. al. Energetic Metabolism and Human Sperm Motility: Impact of CB1 Receptor Activation. Endocrinology, Dec 2010; 151: 5882 - 5892.

- Berasain, C., et al. (2005) J. Biol. Chem. 280: 19012-19020.

- Bhandari, U. et. al. The effect of high-fat diet-induced obesity on cardiovascular toxicity in wistar albino rats. Human and Experimental Toxicology, Sep 2011; 30: 1313 - 1321.

- Byun HS et al (2008) Rheumatology 47: 301-308.

- Caporali, S., et al. (2003) Mol. Biol. Cell 14:5051-5059.

- Carley Glass et. al. MicroRNA-1 transfected embryonic stem cells enhance cardiac myocyte differentiation and inhibit apoptosis by modulating the PTEN/Akt pathway in the infarcted heart. Am J Physiol Heart Circ Physiol, Nov 2011; 301: H2038 - H2049.

- Cartee, L., et al. (2002) Mol. Pharmacol. 61:1313-1321.

- Chakraborty A et al. (2008) PNAS 105: 1134-1139.

- Chen T-H et al (2008) Clin. Cancer Res. 14: 4250- 4258.

- Cheung YY et al. (2007) J. Clin. Invest. 117: 784-793.

- Collin C., et al. (2001) J. Biol. Chem. 276:37602-37611.

- Dai Y et al (2008) Clin. Cancer Res.; 14: 7701 - 7710.

- Daniel, D., et al. (2004) Blood 10.1182/blood-2003-02-0635.

- Datta R., et al. (2000) J. Biol. Chem. 275:31733-31738.

- Ding, H.F., et al. (2000) J. Biol. Chem. 275:38905-38911.

- Doostzadeh J., et al. (2000) J. Biol. Chem. 275:25336-25341.

- Farhana L et al (2009) Mol. Cancer Ther.; 8: 1625 - 1635.

- Feinman, R. et al. HIF-1 Mediates Pathogenic Inflammatory Responses to Intestinal Ischemia-reperfusion Inhury. Am. J. Physiol. Gastrointest. Liver Physiol., 2010; 299: G833-G843

- Fujii T., et al. (2000) J. Biol. Chem. 275:7574-7582.

- Fukutomi, Y. et. al. Apoptosis-Inducing Activity of Clofazimine in Macrophages. Antimicrob. Agents Chemother., Sep 2011; 55: 4000 - 4005.

- Galindo, C.L., et al. (2004) J. Biol. Chem. 279:37597-37612.

- Ghatak S., et al. (2002) J. Biol. Chem. 277:38013-38020.

- Gomez-Angelats M., et al. (2000) J. Biol. Chem. 275:19609-19619.

- Han W et al. (2007) Mol. Cancer Ther. 6: 1641-1649.

- Hojman, P. et. al. Exercise-induced muscle-derived cytokines inhibit mammary cancer cell growth. Am J Physiol Endocrinol Metab, Sep 2011; 301: E504 - E510.

- Hu Y. et al. (2006) J. Immunol. 177: 8522-8530.

- Idogawa M et al (2009) Clin. Cancer Res.; 15: 3725 - 3732.

- Idogawa M et al (2009) Clin. Cancer Res; 15: 3725 - 3732.

and 15-Lipoxygenases. Cancer Prevention Research, Sep 2010; 3: 1132 - 1140.

- Kawakami, M., et al. (2002) Mol. Cancer Thep. 1:999-1007.

- Kenta Moriwaki et al., GDP-mannose-4,6-dehydratase (GMDS) Deficiency Renders Colon Cancer Cells Resistant to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Receptor- and CD95-mediated Apoptosis by Inhibiting Complex II Formation, J. Biol. Chem., Dec 2011; 286: 43123 - 43133.

- Khabbaz KR et al. (2008) J. Thorac. Cardiovasc. Surg. 135: 139 - 146.

- Kim L. and Denkers E.y. (2006) J. cell Sci. 119: 2119-2126.

- Kim SY et al (2008) Blood 111: 5704-5711

- Kim, J. W. et. al. The Role of the 3D Environment in Hypoxia-induced Drug and Apoptosis Resistance. Anticancer Res, Oct 2011; 31: 3237 - 3245.

- Konishi, T., et al. (2005) Mol. Cancer Ther. 4: 743-750.

- Kown, M.H., et al. (2000) Circulation. 102:228-232.

- Lamontagne, L. et al. Hepatits B and Hepatitis C Virus Replication Upregulates Serine Protease Inhibitor Kazal, Resulting in Cellular Resistance to Serine Protease-Dependent Apoptosis. J. Virol. 2010; 84: 907-917

- Lee, S-Y, et al. (2006) Infect. Immun. 10.1128/IAI.01700-06.

- Leu, S. W. et. al. TLR4 through IFN-β Promotes Low Molecular Mass Hyaluronan-Induced Neutrophil Apoptosis. J. Immunol., Jan 2011; 186: 556 - 562.

- Li Y., et al. (2007) Diabetes 56: 656-665.

- Li, C., et al. (2005) J. Biol. Chem. 280: 26193-26199.

- Li, L., et al. (2005) Toxicol. Sci. 86: 116-124.

- Liao, Y. et al. miR-214 Regulates Lactoferrin Expression and Pro-Apoptotic Function in Mammary Epithelial Cells. J. Nutr., Sep 2010; 140: 1552 - 1556.

- Lieman, J.H., et al. (2005) J. Biol. Chem. 280: 10484-10490.

- Macchi, B. et al. The Novel Proapoptotic Actvity of Nonnatural Enantiomer of Lentiginosine. Glycobiology 2010 20: 500-506.

- Machiya J-I et al. (2007) Am. J. Respir. Cell Mol. Biol. 36: 418-426.

- Maity, B. et. al. Regulator of G Protein Signaling 6 (RGS6) Induces Apoptosis via a Mitochondrial-dependent Pathway Not Involving Its GTPase-activating Protein Activity. J. Biol. Chem., Jan 2011; 286: 1409 - 1419.

- Majumdar A. and Du J. (2006) Am. J. Physiol. Gastrointest. Liver Physiol. 290: G49-G-55.

- Malloy PJ et al (2009) Endocrinology; 150: 679 - 686.

- McGaffin KR et al (2009) Cardiovasc Res; 83: 313 - 324.

- McGaffin KR et al (2009) Cardiovasc Res; 83: 313 - 324.

- McGaffin, K. R. et. al. Cardiac-specific leptin receptor deletion exacerbates ischaemic heart failure in mice. Cardiovasc Res, Jan 2011; 89: 60 - 71.

- McGaffin, K. R. et. al. Cardiac-specific leptin receptor deletion exacerbates ischaemic heart failure in mice. Cardiovasc Res, Jan 2011; 89: 60 - 71.

- McGaffin, K. et al. Cardiac-Specific Leptin Receptor Deletion Exacerbates Ischemic Heart Failure in Mice. Cardiovasc. Res., 2010; 10.1093/cvr/cvq288

- Miknyoczki S el al. (2007) Mol. Cancer Ther. 6: 2290 - 2302.

- Misra S et al (2008) J. Biol. Chem. 283: 14335-14344.

- Moriceau S et al (2009) J. Immunol.; 182: 7254 - 7263.

- Nahari D et al. Mol. Cancer Ther. 6: 1329-1337.

- Oh, P. S. et. al. Schlafen-3 decreases cancer stem cell marker expression and autocrine/juxtacrine signaling in FOLFOX-resistant colon cancer cells. Am J Physiol Gastrointest Liver Physiol, Aug 2011; 301: G347 - G355.

- Otogawa K et al. (2008) Am J Physiol Regulatory Integrative Comp Physiol. 294: R311-R320.

- Ph, P. S. et. al. Schlafen-3 decreases cancer stem cell marker expression and autocrine/juxtacrine signaling in FOLFOX-resistant colon cancer cells. Am J Physiol Gastrointest Liver Physiol, Aug 2011; 301: G347 - G355.

- Prokopenko O and Mirochnitchenko O (2009) Am J Physiol Cell Physiol; 296: C1086 - C1097.

- Ramasamy S, et al. Growth Inhibition of Human Gynecologic and Colon Cancer Cells by Phyllanthus watsonii through Apoptosis Induction. PLoS ONE (2012) 7(4): e34793. doi:10.1371/journal.pone.0034793

- Ramlawi B. et al. (2006) Circulation 114: I-257 - I-263.

- Rosato RR et al. (2007) Cancer Res. 67: 9490 - 9500.

- S. A. Bakheet, et. al. Salubrious effects of dexrazoxane against teniposide-induced DNA damage and programmed cell death in murine marrow cells. Mutagenesis, Jul 2011; 26: 533 - 543.

- Saquib, A. Q. et. al. Salubrious effects of dexrazoxane against teniposide-induced DNA damage and programmed cell death in murine marrow cells. Mutagenesis, Mar 2011; 10.1093/mutage/ger013.

- Shain, K.H., et al. (2002) J. Immunol. 168:2544-2553.

- Shiguang Y. et. al. Comparison of sensitivity of Th1, Th2, and Th17 cells to Fas-mediated apoptosisJ. Leukoc. Biol., Mar 2010; 10.1189/jlb.0509352.

- Siddiqui, M. et al. Short-term exposure of 4-hydroxynonenal induces mitochondria-mediated apoptosis in PC12 cells Human and Experimental Toxicology, Apr 2012; 31: 336 - 345.

- Singala DK et al (2008) Am J Physiol Lung Cell Mol Physiol 10.1152/ajplung.00279.2007.

- Singla DK and McDonald DE (2007) Am. J. Physiol Heart Circ. Physiol. 239: H1590-H1595

- Singla DK et al (2008) Am J Physiol Heart Circ. Physiol 295: H907-H913.

- Snow A.L. et al. (2006) J. Immunol. 177: 3283-3293.

- Snow, A.L., et al. (2001) J. Immunol. 167:5404-5411.

- Song, G. et al. MicroRNA-206 Targets Notch3, Activates Apoptosis, and Inhibits Tumor Cell Migration and Focus Formation. J. Biol. Chem. 2009; 284: 31921-31927.

- Su R-Y et al (2008) Mol. Cancer Ther.; 7: 3429 - 3440.

- Suh, K.S., et al. (2004) J. Biol. Chem. 279:4632-4641.

- Sun HL et al (2009) Clin. Cancer Res.; 15: 4904 - 4914.

- Tian F et al (2009) Blood; 113: 5352 - 5360.

- Toyonaga, J. et. al. Spironolactone inhibits hyperglycemia-induced podocyte injury by attenuating ROS production. Nephrol. Dial. Transplant., Jan 2011; 10.1093/ndt/gfq750.

- Toyonaga, J. et. al. Spironolactone inhibits hyperglycemia-induced podocyte injury by attenuating ROS production. Nephrol. Dial. Transplant., Aug 2011; 26: 2475 - 2484.

- Toyonaga, J. et. al. Spironolactone inhibits hyperglycemia-induced podocyte injury by attenuating ROS production. Nephrol. Dial. Transplant., Aug 2011; 26: 2475 - 2484.

- Vu, C.C.Q., et al. (2001) J. Biol. Chem. 276:37602-37611.

- Watkins, A. et. al. Hypoxia-inducible factor plays a gut-injurious role in intestinal ischemia reperfusion injury. Am J Physiol Gastrointest Liver Physiol, May 2011; 300: G853 - G861.

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- Williamson CL et al (2008) Am J Physiol Heart Circ Physiol 294: H249-H256.

- Witko-Sarsat, V. et. al. Proliferating cell nuclear antigen acts as a cytoplasmic platform controlling human neutrophil survival. J. Exp. Med., Nov 2010; 207: 2631 - 2645.

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- Yuan, S. Y. et. al. Comparative Nephrotoxicity of Aristolochic Acid and Tetrandrine In Vitro and In Vivo. International Journal of Toxicology, Feb 2011; 30: 35 - 46.


- Yujiang Fang, et. al. A Possible Role for Perforin and Granzyme B in Resveratrol Enhanced Radiosensitivity of Prostate Cancer.J Androl, Nov 2011; 10.2164/jandrol.111.015164.

- Zauli, G. et. al. Dasatinib Plus Nutlin-3 Shows Synergistic Antileukemic Activity in Both p53wild-type and p53mutated B Chronic Lymphocytic Leukemias by Inhibiting the Akt Pathway. Clin. Cancer Res., Feb 2011; 17: 762 - 770.

- Zhang Y. et. al. Nuclear Receptor SHP, a Death Receptor That Targets Mitochondria, Induces Apoptosis and Inhibits Tumor Growth Mol. Cell. Biol., Mar 2010; 30: 1341 - 1356.

- Zhiang, Y. et al. Nuclear Receptor SHP, a Death Receptor that Targets Mitochondria, Induces Apoptosis and Inhibits Tumor Growth. Mol. Cell. Biol. 2010; 10.1128/MCB.01076-09.

- Zhong, Z. et.al Protein S Protects Neurons from Excitotoxic Injury by Activating the TAM Receptor Tyro3–Phosphatidylinositol 3-Kinase–Akt Pathway through Its Sex Hormone-Binding Globulin-Like Region.. J. Neurosci., Nov 2010; 30: 15521 - 15534.

- Zhonghua Li, et al., Critical Role for Voltage-Dependent Anion Channel 2 in Infectious Bursal Disease Virus-Induced Apoptosis in Host Cells via Interaction with VP5, J. Virol., Feb 2012; 86: 1328 - 1338.

- Zhou F. et al. (2006) Mol. Cell Biol. 26: 3478-3491.

- Zhuang S. et al. (2006) Am. J. Physiol. Renal Physiol. 10.1152/ajprenal.00170.2006.

- Jun, H. et al. Lack of Glucose Recycling between Endoplasmic Reticulum and Cytoplasm Underlies Cellular Dysfunction in Glucose-6-Phosphatase--Deficient Neutrophils in a Congenital Neutropenia Syndrome. Blood, 2010; 116: 2783-2792

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Answer

Thank you for contacting us. I have answered each of your questions below. Please let me know if you have any additional questions about this or any Abcam product.

1. Does this kit recognize rat caspase-3?

This will recognize all mammalian forms (90% homology of Caspase 3 w/rat)



2. Does this kit recognize both activated and non-activated caspase-3 or either?

The assay is based on spectrophotometric detection of thechromophore p-nitroaniline (p-NA) after cleavage from the labeledsubstrate DEVD-p-NA. Therefore it should only recognize activated Caspase 3.



3. Can I use this kit for tissue Caspase 3 assay? If so, a 50-200 mcg protein use remains same as a recommended protocol?

Yes, you can use tissue lysates for this kit. Use exactly the same protocol. Use 200-400 µl of the lysis buffer for ach 10 mg of tissue. Samples should be completely homogenized, preferably using a Dounce homogenizer. Some substances can interfere with the assays and should be avoided in sample preparation. Please see individual kit datasheets for more specific information on which substances may affect the specific kits, but in general, substances such as EDTA (> 0.5mM), ascorbic acid (> 0.2%), sodium azide (> 0.2%), NP-40 and Tween-20 (>1%) are known to interfere with these assays and should be avoided in sample preparation. Moreover, we recommend deproteinating the sample prior analysis using a 10kD spin column (ab93349).



4. for tissue assay, any additional suggestions on the protocol?

Use exactly the same protocol. Use 200-400 µl of the lysis buffer for ach 10 mg of tissue.






I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting Abcam.

The dilution buffer is to dilute the final samples before reading their absorbance, in case of the undiluted readings being above the detection range of the instrument.
Hope this information has been helpful for you.
Please let me know if you have any other questions.

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Answer


The dilution buffer is used in case you do not have a plate reader an need to read the concentration manually in a cuvette: Step10. "Read samples at 400 or 405 nm in a microtiter plate reader, or
spectrophotometer using a 100 μl micro-quartz cuvette, or dilute
sample to 1 ml with Dilution Buffer and using regular cuvette."

In step 7, you are correct, the protein amount in the samples is measured and than adjusted with the lysis buffer for the assay.

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1-10 of 12 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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